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Metabolic Engineering of Escherichia coli for Enhanced Production of ent-Kaurenoic Acid
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 이평천 | - |
| dc.contributor.author | 오순환 | - |
| dc.date.accessioned | 2018-11-08T08:10:58Z | - |
| dc.date.available | 2018-11-08T08:10:58Z | - |
| dc.date.issued | 2017-02 | - |
| dc.identifier.other | 24349 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/11293 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :분자과학기술학과,2017. 2 | - |
| dc.description.tableofcontents | CONTENTS ABSTRACT i LIST OF TABLES iv TABLE OF FIGURES v LIST OF ABBREVIATIONS vi 1. Introduction - 1 - 2. Meterials & Methods - 5 - 2.1 Plasmids Construction. - 5 - 2.2 Construction of linear DNA fragment for genome editing. - 5 - 2.3 Genome Engineering. - 6 - 2.4 Media and Culture Condition. - 7 - 2.5 Culture condition of bioreactor. - 7 - 2.6 Quantitative Real-Time PCR. - 7 - 2.7 Product Extraction and Analytical Methods. - 8 - 2.8 Site Directed Mutagenesis (SDM). - 9 - 2.9 SDS-PAGE and Western Blot. - 9 - 3. Results - 17 - 3.1 Construction of Genome Edited Strain. - 17 - 3.2 Growth and ent-Kaurenoic Acid Production Analysis of Recombinant Strains - 19 - 3.3 Scale-up Cultivation to compare ent-Kaurenoic Production between Recombinant Strains. - 21 - 3.4 Quantification of RNA Levels of Precursor Pathway Genes - 23 - 3.5 Construction of KO Mutants and Products Profiling. - 25 - 3.6 Quantification of Protein Expression Level of KO - 29 - 3.7 Scale-up Cultivation to Compare between Wild Type and S44T Mutant. - 31 - 4. Discussion - 33 - 5. References - 36 - ABSTRACT IN KOREAN - 39 - ACKNOWLEDGEMENTS - 40 - | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Metabolic Engineering of Escherichia coli for Enhanced Production of ent-Kaurenoic Acid | - |
| dc.title.alternative | Soon Hwan Oh | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Soon Hwan Oh | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2017. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 770253 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000024349 | - |
| dc.subject.keyword | ent-kaurenoic acid | - |
| dc.subject.keyword | metabolic engineering | - |
| dc.subject.keyword | Escherichia coli | - |
| dc.subject.keyword | ent-kaurene | - |
| dc.subject.keyword | genome editing | - |
| dc.description.alternativeAbstract | Plant derived terpenoid compounds are used as aromatic and pharmacological agents and they are industrially valuable materials. The ent-kaurenoic acid, a kauranic diterpenoid, and its derivatives have been shown to have a variety of pharmacological activities and the interest in their utilization is increasing. In particular, steviol glycosides derived from ent-kaurenoids are highly sweet and non-caloric natural products, which are valuable in the food and beverage industry. In order to enhance the biosynthetic pathway of ent-kaurenoic acid in E. coli, genome editing techniques were used to insert precursor related genes into the E. coli genomic DNA and induce stable overexpression. The production of ent-kaurenoic acid in the obtained genome edited strain increased 6.3 times compared to the non-overexpressed strain and 1.9 times compared to the overexpressed strain using plasmid system. The gene transcription levels between over-expression systems were compared by quantitative polymerase chain reaction (qPCR) and the transcription level of genome insertion system was lower than that of plasmid system. The ent-kaurene oxidase (KO) wild-type and three mutants were tested in flasks and 1.5 L bioreactor culture. The ent-kaurenoic acid production in the S44T mutant increased 2.2-fold and 1.2-fold in each experiment compared to the wild-type. The results of this study could be used as a basis for the development of microbial biosynthetic strains of other terpenoid compounds as well as ent-kaurenoids and various derivatives. | - |
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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