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FCS 및 ECH를 이용한 재조합 대장균에서 L-타이로신으로부터 페놀알데히드 및 멜라닌 색소의 생산
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 최권영 | - |
| dc.contributor.author | 장세영 | - |
| dc.date.accessioned | 2018-11-08T08:11:49Z | - |
| dc.date.available | 2018-11-08T08:11:49Z | - |
| dc.date.issued | 2018-02 | - |
| dc.identifier.other | 27499 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/11538 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :환경안전공학과,2018. 2 | - |
| dc.description.abstract | 본 연구에서는 TAL (tyrosine ammonia-lyase), C3H (p-coumarate 3-hydroxylase), FCS (feruloyl-CoA synthetase) 및 ECH (enoyl-CoA hydratase)를 비롯한 L-타이로신 변환 효소를 발현하도록 대장균을 조작하였다. protocatechualdehyde와 p-hydroxybenzaldehyde 생산 경로로 구성된 이화 회로는 개별 효소의 조합 발현을 통해 재구성되었다. 첫째, FCS와 ECH를 발현하는 세포는 각각 5 mM의 카페익산과 페룰산을 기질로하여 protocatechualdehyde (70.5 %)와 vanillin (96.5 %)로 전환시킬 수 있다. 둘째, TAL과 C3H는 FCS와 ECH와 함께 발현되었다. 이 균주는 L-타이로신을 카페익산으로 전환시킨 다음 protocatechualdehyde로 전환시키는 것을 목표로 하였다. 실험 결과 p-coumarate 3-hydroxylase 과정을 생략하여 생성되는 p-hydroxybenzaldehyde가 protocatechualdehyde와 동시에 생성됨을 알게되었다. 또한, Ascorbic acid는 catechol aldehyde계 멜라닌 생성 억제제로 사용되었으며 protocatechualdehyde와 p-hydroxybenzaldehyde의 생산 수율은 각각 31.0 ± 5.6, 24.0 ± 4.2 mg / L이었다. 마지막으로 카페인 산의 존재 하에서 FCS와 ECH를 동시에 발현시킴으로써 40.9 ± 6.2 mg / L / h의 높은 생산 속도로 카페익산 기반의 멜라닌이 관찰되었다. | - |
| dc.description.tableofcontents | 1. Introduction 1 2. Meterials and Methods 6 2.1. Bacterial strains and chemicals 6 2.2 Cloning and expression of TAL, C3H, FCS and ECH. 6 2.3 Whole-cell production of vanillin, p-coumaric acid, p-hydroxybenzaldehyde, and protocatechualdehyde 9 2.4. HPLC analysis of products generated 9 2.5. Quantification of melanin generated 10 2.6. Cloning and expression of MelC and CadA 10 2.7. Whole-cell production of melanin 11 2.8. Preparation of melanin 12 3. Results and discussion 13 3.1. Plasmid construction and gene expression in E. coli 13 3.2. Module I: Conversion of L-tyrosine to p-coumaric acid and caffeic acid 13 3.3. Module II: Conversion of ferulic acid to vanillin 14 3.4. Module II: Conversion of caffeic acid into protocatechualdehyde and melanin 17 3.5. Module I and II: Conversion of L-tyrosine to p-hydroxybenzaldehyde and protocatechualdehyde 19 3.6. Production of melanin from protocatechualdehyde and caffeic acid 21 3.7. Pathway construction and gene expression in E. coli 23 3.8. Production of melanin pigment 25 4. Conclusions 29 5. References 32 6. Supplementary Data 37 7. Abstracts in Korean 43 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | FCS 및 ECH를 이용한 재조합 대장균에서 L-타이로신으로부터 페놀알데히드 및 멜라닌 색소의 생산 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 환경안전공학과 | - |
| dc.date.awarded | 2018. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 800430 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000027499 | - |
| dc.subject.keyword | 멜라닌 | - |
| dc.subject.keyword | protocatechualdehyde | - |
| dc.description.alternativeAbstract | In this study, we engineered E. coli cells to express L-tyrosine converting enzymes, including tyrosine ammonia-lyase (TAL), p-coumarate 3-hydroxylase (C3H), feruloyl-CoA synthetase (FCS), and enoyl-CoA hydratase/aldolase (ECH). A catabolic circuit, which consisted of the protocatechualdehyde and p-hydroxybenzaldehyde production pathways, was reconstituted through combinatorial expression of discrete enzymes. First, cells expressing FCS and ECH could convert each 5 mM of caffeic acid and ferulic acid into protocatechualdehyde (70.5%) and vanillin (96.5%), respectively. Second, TAL and C3H were co-expressed with FCS and ECH. This strain converted L-tyrosine into caffeic acid, which was then converted into protocatechualdehyde. Ascorbic acid was used as an inhibitor of catechol aldehyde-based melanin formation, and the production yields of protocatechualdehyde and p-hydroxybenzaldehyde were 31.0 ± 5.6 and 24.0 ± 4.2 mg/L, respectively. Finally, caffeic acid-based melanin formation was observed with higher production rate of 40.9 ± 6.2 mg/L/h by co-expressing FCS and ECH in the presence of caffeic acid. | - |
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