Microbial production of retinoic acid using metabolically engineered Escherichia coli
- 주제(키워드) retinoid , retinoic acid , metabolic engineering , retinaldehyde dehydrogenase , β-carotene 15 , 15’-oxygenase
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 이평천
- 발행년도 2021
- 학위수여년월 2021. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031205
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Microbial production of bioactive retinoids including retinol and retinyl ester have been successfully reported. Nonetheless, there is no report of the microbial biosynthesis of retinoic acid. Therefore, two genes (blhSR and raldhHS) encoding retinoic acid biosynthesis enzymes [β-carotene 15,15’-oxygenase (Blh) and retinaldehyde dehydrogenase2 (RALDH2)] were synthetically redesigned to be modularly expressed. Co-expression of the blhSR and raldhHS genes on the plasmid system in the metabolically engineered β-carotene-producing Escherichia coli strain produced 0.59 ± 0.06 mg/L of retinoic acid in flask cultivation. Deletion of an ybbO gene encoding promiscuous aldehyde reductase enhanced 2.4-fold increase in retinoic acid from 0.59 ± 0.06 mg/L to 1.43 ± 0.06 mg/L. Engineering of the 5’UTR sequence of the blhSR and raldhHS genes future enhanced retinoic acid titer from 1.43 ± 0.06 mg/L to 3.46 ± 0.16 mg/L. Finally, a batch culture operated under the conditions (37°C, pH 7.0, and 50% DO) enhanced up to 8.20 ± 0.27 mg/L of retinoic acid in a bioreactor. As the construction and culture of the retinoic acid-producing strain carried out in this study is at an early stage in the development of the microbial process for retinoic acid, further optimization of the expression level of the retinoic acid pathway genes, protein engineering of Blh and RALDH2, and the fermentation operation and media optimization should synergistically increase the current titer of retinoic acid in E.coli.
more목차
1.Introduction 1
2. Materials and Methods 5
2.1 Strains, media, and culture conditions. 5
2.2 Plasmid construction for the expression of retinoid biosynthesis pathway enzymes. 5
2.3 Deletion of ybbO gene in BETA-1 strain. 6
2.4 Integrating two retinoic acid pathway genes into BETA-1ΔybbO strain. 7
2.5 Reverse-Transcription PCR (RT- PCR). 8
2.6 SDS-PAGE and Western Blot analysis. 8
2.7 Bioreactor fermentation. 9
2.8 Extraction of retinoids. 9
2.9 Analysis of HPLC and Mass Spectrometry(MS). 10
3. Results 15
3.1 Expression of retinoic acid pathway enzyme. 15
3.2 Construction of retinoic acid biosynthetic pathway in BETA-1 strain. 17
3.3 Enhanced retinoic acid production by deletion of ybbO gene in BETA-1 strain. 20
3.4 Transcription control engineering of blhSR and raldhHS genes for improving retinoic acid production. 22
3.5 Construction and expression of retinoic acid-biosynthetic pathway genes on the genome of RA1 strain. 25
3.6 Bioreactor study of retinoic acid-producing strains. 29
4.Discussion 31
5.References 34
