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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林曉青(Hsiao-Ching Lin) | |
| dc.contributor.author | Li-Xun Chen | en |
| dc.contributor.author | 陳立訓 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:50:20Z | - |
| dc.date.available | 2025-08-17 | |
| dc.date.copyright | 2020-09-17 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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Journal of the American Chemical Society 2019, 141 (20), 8068-8072. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69071 | - |
| dc.description.abstract | 在細菌與真菌中，非核糖體胜肽合成酶為催化生成非核糖體胜肽之酵素。棘孢素是由棘孢麴黴所生產的萜烯胺基酸天然雜合物。先前的研究中發現 ane 基因群負責棘孢素的生物合成。然而，對於下列兩個步驟中的重要酵素及其機制仍尚未了解：(1) 倍半萜類的骨架轉換為十四碳倍半萜類之去甲基化反應。(2) 參與在轉移脯胺酸形成萜烯胺基酸雜合物反應中，非核糖體肽合成酶 AneB和水解酶 AneE 之催化功能。 為了了解倍半萜類去甲基化反應，我們使用米麴菌作為異源表達系統，並重建三個細胞色素 P450s−AneD、AneF 以及 AneG之功能。結果發現這三個細胞色素 P450s 的接續催化能誘發十五碳倍半萜類的去甲基化，進而生成十四碳倍半萜產物。為了鑑定 AneB 的功能，我們以突變以及表達部分片段 AneB 以進行生物轉化試驗。實驗結果確認 AneB 催化功能的關鍵胺基酸位點，並藉此提出萜烯胺基酸的合成機轉。另外，藉由體外以及體內試驗，我們證明了水解酶 AneE 具有促進非核糖體肽合成酶催化功能的效果。此外，比起野生型的菌株，我們發現在 AneE 基因敲除的棘孢麴黴菌株中有兩種二次代謝物無法被生產，這表示 AneE 可能參與其生物合成步驟。我們成功分離並鑑定此兩種代謝物麥角黃酮酸 D 和 F，但水解酶在此途徑扮演的角色仍需進一步的探討。 在本次研究當中，我們發現十四碳倍半萜類的生成機制，並進一步鑑定了非核糖體胜肽合成酶和水解酶的催化特性。此研究擴增了棘孢素的生合成途徑，並使得生物體生產萜烯胺基酸之天然物的方式更被為了解。 | zh_TW |
| dc.description.abstract | Non-ribosomal peptide synthetases (NRPSs) catalyze the formation of backbone of non-ribosomal peptides (NRPs) in many bacteria and fungi. Aculenes are amino acid-terpene hybrid natural products produced by Aspergillus aculeatus. In the previous study, the ane gene cluster that is responsible for the biosynthesis of aculenes has been identified. However, the enzymes and their mechanisms involved in the two key steps to form aculenes remained unknown, (i) demethylation process to convert the sesquiterpene (C15) core skeleton to the norsesquiterpene (C14); (ii) the function of the hydrolase (AneE) and the NRPS (AneB) involved in proline transfer to form the terpene-amino acid hybrid products. To understand the demethylation process from the sesquiterpene skeleton, we have reconstituted three cytochrome P450s in the Aspergillus oryzae heterologous system. The results showed that three cytochrome P450s (AneD, AneF, and AneG) are required and catalyze a stepwise demethylation process. To characterize the function of AneB (NRPS), the truncated and mutated AneB have been generated. Based on the results of the biotransformation experiments, several key residues in AneB were discovered and the catalytic mechanism was proposed. In both of in vitro and in vivo experiments, the results supported that the role of AneE (hydrolase) to facilitate the formation of terpene-amino acid product catalyzed by AneB. On the other hand, we observed that AneE may take part in the biosynthesis of other two secondary metabolites from A. aculeatus due to their disappearance in the AneE deleted mutant compared to the wild type strain. Those two metabolites have been purified and elucidated as secalonic acids D and F. The role of AneE involved in the biosynthesis of secalonic acids will be further investigated. Our study elucidated the key steps in the formation of norsesquiterpenes and characterized the biochemical properties of the NRPS and hydrolase in aculenes biosynthesis. The study expanded the biosynthetic pathway of aculenes and led to understand how nature generates amino acid-terpene hybrid natural products | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:50:20Z (GMT). No. of bitstreams: 1 U0001-1608202023343700.pdf: 8487932 bytes, checksum: 78279030185f725b809e6b06aaa5881f (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 謝辭 i 中文摘要 ii Abstract iv Table of Contents vi List of Figures xi List of Schemes xv List of Tables xvi Abbreviations xvii 1. Introduction 1 1.1 Non-ribosomal peptide synthetases (NRPSs) 1 1.2 Product release mechanism 5 1.2.1 TE domain performs hydrolysis or macro-cyclization 5 1.2.2 C domain mediates product release 6 1.2.3 Reductive domain (R domain) catalyzes reductive release 7 1.2.4 Ketosynthase (KS) domain carries out Dieckmann cyclization 7 1.3 The biosynthesis of hybrid-type NPs 10 1.3.1 Polyketide-nonribosomal peptide (PK-NRP) hybrid NPs 10 1.3.2 Polyketide-terpene (PK-TC) hybrid NPs 11 1.4 Aculenes and Terpene-amino acid Hybrids 14 1.4.1 Aspergillus aculeatus and aculenes biosynthetic pathway 14 1.4.2 The role of AneB and AneE in aculene biosynthetic pathway 15 1.5 Aim of study 17 2. Materials and Method 18 2.1 Strain, medium, and cultivation condition 18 2.2 General chemical method 19 2.3 General Molecular biology experiment 20 2.4 Construction of plasmid 24 2.4.1 Construction of plasmids for the heterologous expression in S. cerevisiae 24 2.4.2 Construction of plasmids of aneC-aneF-xw55, aneD-xw02, and aneG-xw06 for the heterologous expression in S. cerevisiae 24 2.4.3 Construction of plasmids of aneB-xw55, aneB-S596A-xw55 or aneB-C-xw55, aneE-xw06 for purification of AneB and AneE from S. cerevisiae 25 2.4.4 Construction of plasmids of aneB-A-xw55, aneB-T-xw55, aneB-C-xw55, and aneB-AT-xw55 for biotrasformation of S. cerevisiae 25 2.4.5 Construction of plasmids for biotrasformation of S. cerevisiae expressing mutant aneB 26 2.4.6 Construction of plasmids of AneC-pAdeA, aneF-pPTRI, AneC-aneF-pAdeA, AneD-pTAex3, and AneG-pPTRI for Aspergillus oryzae transformation 27 2.4.7 Construction of plasmids of aneB, aneB-A domain, aneB-T domain, aneB-C domain, and aneB-S596A for purification of AneB from Escherichia coli 28 2.5 Heterologous reconstitution of aculenes upstream pathway by Saccharomyces cerevisiae 29 2.6 Heterologous reconstitution of aculenes upstream pathway by Aspergillus oryzae 30 2.7. Protein Purification from S. cerevisiae or Escherichia coli 32 2.7.1 Expression and Purification of AneB and AneE from S. cerevisiae 32 2.7.2 Expression and Purification of AneB from Escherichia coli 32 2.7.3 Expression and purification of His6 or His10-tagged protein 34 2.7.4 Expression and Purification of AneB from E. coli by anti-FLAG M2 resin 34 2.8 Biotransformation of S. cerevisiae expressing aneB 35 2.8.1 Biotrasformation of S. cerevisiae expressing A domain, T domain, C domain, or A-T di-domain of aneB 35 2.8.2 Biotransformation of S. cerevisiae expressing mutant aneB 35 2.8.3 Biotransformation of S. cerevisiae expressing aneBE 36 2.9 In vitro assay of AneB and AneE 37 2.10 Chemical complementation in Aspergillus aculeatus 37 2.11.1 Isolation and purification of aculene D (4), asperaculane G (13) from Aspergillus oryzae 38 2.11.2 Isolation and purification of asperaculane H (14) from S. cerevisiae 40 2.11.3 Isolation and purification of secalonic acid D (15) and secalonic acid F (16) from A. aculeatus 42 3. Result and Discussion 43 3.1.1 The demethylation process to form norsesquiterpene 43 3.1.2 Heterologous expression of upstream pathway of aculenes in S. cerevisiae 45 3.1.3 Reconstitution of aculene biosynthetic pathway in Aspergillus oryzae 48 3.1.4 Isolation and purification of asperaculane G (13) from Aspergillus oryzae expressing aneCDFG 52 3.1.5 Chemical complementation of asperaculane G (13) in A. aculeatus mutants 55 3.2 Isolation and purification of asperaculnae H (14) from S. cerevisiae 57 3.3 The expand aculene biosynthetic pathway 58 3.4 Bioinformatic analysis of the domains and catalytic motifs of AneB 61 3.4.1 Purification of the truncated and mutanted AneB proteins 63 3.4.2 Biotransformation of S. cerevisiae expressing truncated aneB supplemented by aculene D 66 3.4.3 Biotransformation of S. cerevisiae expressing mutanted aneB supplemented by aculene D 68 3.5.1 The role of AneE in the aculene biosynthetic pathway 70 3.5.2 Biotransformation of S. cerevisiae expressing aneB and aneE 71 3.5.3 In vitro assay of AneB and AneE 73 3.6 The catalytic mechanism of the terpene-amino acid formation 75 3.7 AneE hydrolase may participate in other secondary metabolite biosynthesis 77 3.7.1 Isolation and purification of secalonic acid D (15) from ∆aneD mutant of A. aucleatus 77 3.7.2 Isolation and purification of secalonic acid F (16) from ∆aneD mutant of A. aucleatus 79 3.7.3 The role of AneA and AneE in the secalonic acid biosynthesis 82 4. Conclusion 86 5. Reference 87 6. Appendix 91 | |
| dc.language.iso | en | |
| dc.subject | 非核糖體肽合成酶 | zh_TW |
| dc.subject | 細胞色素 P450 | zh_TW |
| dc.subject | 去甲基化 | zh_TW |
| dc.subject | 萜烯胺基酸 | zh_TW |
| dc.subject | 非核糖體肽合成酶 | zh_TW |
| dc.subject | 水解酶 | zh_TW |
| dc.subject | 水解酶 | zh_TW |
| dc.subject | 細胞色素 P450 | zh_TW |
| dc.subject | 去甲基化 | zh_TW |
| dc.subject | 萜烯胺基酸 | zh_TW |
| dc.subject | Terpene-amino acid hybrid | en |
| dc.subject | demethylation | en |
| dc.subject | hydrolase | en |
| dc.subject | non-ribosomal peptide synthetase | en |
| dc.subject | cytochrome P450 | en |
| dc.title | 解析棘孢素生物合成途徑中形成萜烯胺基酸之酶工具 | zh_TW |
| dc.title | Characterization of the Enzymatic Machineries for the Formation of Terpene-Amino acid Hybrid Compounds in Aculenes Biosynthesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳世雄(Shih-Hsiung Wu),林俊宏(Chun-Hung Lin),李宗璘(Tsung-Lin Li) | |
| dc.subject.keyword | 萜烯胺基酸,去甲基化,細胞色素 P450,水解酶,非核糖體肽合成酶, | zh_TW |
| dc.subject.keyword | Terpene-amino acid hybrid,demethylation,cytochrome P450,hydrolase,non-ribosomal peptide synthetase, | en |
| dc.relation.page | 127 | |
| dc.identifier.doi | 10.6342/NTU202003636 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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| U0001-1608202023343700.pdf 未授權公開取用 | 8.29 MB | Adobe PDF |
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