Metabolic Engineering of Escherichia coli for Enhanced Production of ent-Kaurenoic Acid
Metabolic Engineering of Escherichia coli for Enhanced Production of ent-Kaurenoic Acid
- 주제(키워드) ent-kaurenoic acid , metabolic engineering , Escherichia coli , ent-kaurene , genome editing
- 발행기관 아주대학교
- 지도교수 이평천
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024349
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
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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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 -
