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스테비올 과생산을 위한 대장균 대사공학
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 이평천 | - |
| dc.contributor.author | 문준호 | - |
| dc.date.accessioned | 2019-04-01T16:40:20Z | - |
| dc.date.available | 2019-04-01T16:40:20Z | - |
| dc.date.issued | 2019--2 | - |
| dc.identifier.other | 28311 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/14851 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :분자과학기술학과,2019. 2 | - |
| dc.description.tableofcontents | 1. Introduction……………………………………………………………………..1 2. Materials and Methods…………………………………………………………6 2.1 Strains, plasmids, and culture conditions…………………………………….6 2.2 Construction of plasmids……………………………………………………..6 2.3 Integration of ent-kaurene pathway into E.coli by CRISPR/Cas9………...…7 2.4 Construction of AtKO N-terminal mutant and SDS-PAGE analysis….……...8 2.5 Genome engineering for improving NADPH availability and measurements of NADP+ and NADPH concentrations…………………………...……………..9 2.6 Construction of artificial self-sufficient trCYP714A2-AtCPR2 fusion protein.9 2.7 Condition of batch fermentation. ……………………………………………10 2.8 Extraction and analysis of products. …………………………………….…10 3. Results………………………………………………………………………….19 3.1 Construction of plasmid-free strain for constitutive production of ent-kaurene……………………………………………………………………..19 3.2 The effect of 5’-UTR on ent-kaurene production…………….…………...…20 3.3 Comparison of kaurene oxidase from S. rebaudiana and A. thaliana for ent-kaurenoic acid production in E.coli……………………………………….…23 3.4 Effect of NADPH/NADP+ ratio on production of ent-kaurenoic acid and stevio………………………………………………………………………...25 3.5 Effects of fusion proteins on steviol production…………………………..…27 3.6 Production of steviol through batch fermentation…………………………...29 4. Discussion………………………………………………………………………31 5. References………………………………………………………………...……33 ABSTRACT IN KOREAN………………………………………………………37 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 스테비올 과생산을 위한 대장균 대사공학 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2019. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 905467 | - |
| dc.identifier.uci | I804:41038-000000028311 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000028311 | - |
| dc.description.alternativeAbstract | As the incidence of diseases, such as diabetes and obesity, increases, there is an increasing global demand for natural sweeteners to replace sugar. Steviol glycosides, compounds extracted from the herbal plant stevia rebaudiana Bertoni, are valuable in the food and beverage industry because they have intense sweetness and no calories. Recently, engineered Escherichia coli (E.coli) was developed to produce steviol glycosides. However, the conversion rate of ent-kaurenoic acid to steviol, which is aglycone of steviol glycosides, was low due to difficulty of functional expression of cytochrome P450 (P450) in E.coli. In this study, to enhance the biosynthesis of steviol in E. coli, engineering of the P450 proteins was carried out, and genomic editing technology was used, to improve NADPH availability. These enhancements produced approximately 4.3 times more steviol than a control in batch fermentation. In addition, the world’s first ent-kaurene biosynthesis pathway was inserted into the E.coli chromosome to reduce the metabolic burden from plasmids. This constructed strain can produce ent-kaurene without adding antibiotics and inducers. The results of this study can be used to develop biosynthetic strains not only steviol but also other P450-derived metabolites. | - |
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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