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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林曉青(Hsiao-Ching Lin) | |
| dc.contributor.author | Chi-Fang Lee | en |
| dc.contributor.author | 李奇芳 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:59:29Z | - |
| dc.date.available | 2028-12-31 | |
| dc.date.copyright | 2018-08-01 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-26 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71223 | - |
| dc.description.abstract | Aculenes are sesquiterpenes produced by Aspergillus aculeatus, a black filamentous fungus. Among the identified aculenes, aculenes A-D and asperaculene B contain an unprecedented norsesquiterpene scaffold that may derive from ent-daucane-type skeleton. As a cumulative number of aculenes that has been identified with chemical investigation, the genetic and enzymatic basis involved aculenes biosynthesis remains unknown. In this study, we have identified the biosynthetic gene cluster of aculenes and proposed the biosynthetic pathway based on gene inactivation, heterologous reconstitution, and biochemical characterization.
From knockout strains and heterologous co-expression transformants of saccharomyces cerevisiae, we isolated thirteen aculene derivatives including seven new compounds. Based on the results, we discovered three P450 monooxygenases involving in multiple oxidative steps, one dioxygenase that introducing a double bond, and one non-ribosomal peptide synthase (NRPS) that transferring a proline moiety to the sesquiterpene skeleton. Furthermore, we characterized a terpene synthase that forms the ent-daucane-type skeleton of aculenes with heterologous expression. Biochemical characterization of the NRPS and hydrolase were also investigated. Interestingly, the deletion of a gene coding proline iminopeptidase on the aculene biosynthetic gene cluster did not influence the production of aculene A. The bioinformatic analysis on the A. aculeatus genome indicated that there are two proline iminopeptidase homologs within the genome, while most fungi only have one proline iminopeptidase. We hypothesized that the aculene-type metabolites may play a certain role in the interaction with the two proline iminopeptidase homologs. Further molecule-enzyme interaction will be investigated by enzymatic assay methods. We will continue to characterize the function of three P450s and to identify the key enzymes involved in the generation of norsesquiterpene skeleton. Furthermore, biological implication may be obtained based on the investigation of the interaction between aculene derivatives and proline iminopeptidase homologs. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:59:29Z (GMT). No. of bitstreams: 1 ntu-107-R05b46011-1.pdf: 14677016 bytes, checksum: c32cea5554705e9b9a0dd546e3588411 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii Table of Contents I List of Figures III List of Tables VI List of Schemes VII Abbreviations VIII 1 Introduction 1 1.1 Terpenoid 1 1.2 Aspergillus aculeatus and aculenes 4 1.3 Proline iminopeptidase 7 1.4 Duplicated target 8 1.5 Aim of this study 9 2 Materials and Method 10 2.1 Strains, media and growth conditions 10 2.2 Molecular biology experiments 10 2.3 Chemical analysis 11 2.4 Cloning and plasmid construction 12 2.4.1 Knockout cassettes 16 2.4.2 Cloning of pXW55-AneC, pXW02-AneD, pXW06-AneF, pXW55-AneG and pXW55-AneC-AneG for heterologous expression in Saccharomyces cerevisiae 16 2.4.3 Cloning of pET28-AneC, pET28-AaPIP, pET28-AneG, pXW55-AneB and pXW06-AneE for protein purification 17 2.5 A. aculeatus protoplasting, transformation and gene deletion 17 2.5.1 Protoplasting 17 2.5.2 sgRNA and Cas9:guide RNA ribonucleoprotein (Cas9 RNP) preparation 18 2.5.3 Fungal transformation 19 2.6 Heterologous reconstitution of biosynthesis in Saccharomyces cerevisiae 19 2.7 Metabolites isolation 20 2.7.1 Purification of aculene A (1), B (2), C (3), D (3) from wild type A. aculeatus 20 2.7.2 Purification of aculene B (2) and compound 13 fromΔaneA mutant 21 2.7.3 Purification of compound 8 and compound 9 fromΔaneD mutant 22 2.7.4 Purification of compound 6 fromΔaneE mutant 23 2.7.5 Purification of compound 7 fromΔaneF mutant 23 2.7.6 Purification of compound 5 and 9 fromΔaneG mutant 24 2.7.7 Purification of compound 10 from AneC and AneF co-expression transformant 25 2.7.8 Purification of compound 11 from AneC, AneD and AneF co-expression transformant 25 2.7.9 Purification of compound 12 from AneC, AneF and AneG co-expression transformant 26 2.8 Expression and purification of His6-tagged protein 40 2.8.1 Expression of AneC, AaPIP, AneH in E. coli 40 2.8.2 Expression of AneB, AneE in S. cerevisiae 40 2.9 Purification of His6-tagged protein 41 2.10 In vitro assay 42 2.10.1 AneC-terpene synthase 42 2.10.2 NRPS and hyhrolase 42 2.11 Enzymatic assay of proline iminopeptidase 42 3 Results and Discussion 43 3.1 Biosynthetic gene cluster and gene inactivation 43 3.2 Aculene-type compounds with C14-OH are shunt product 48 3.3 Heterologous reconstitution of biosynthesis in yeast 49 3.4 Structure elucidation 51 3.4.1 Compound 1-6 51 3.4.2 Compound 7 59 3.4.3 Compound 8 61 3.4.4 Compound 9 63 3.4.5 Compound 10 66 3.4.6 Compounds 11 and 12 68 3.4.7 Compound 13 72 3.5 AneD and AneG may be involved in multiple oxidation and decarboxylation steps 74 3.6 Biochemical characterization of AneB, AneC and AneE 76 3.6.1 In vitro assay of AneC 76 3.6.2 In vitro assay of AneB and AneE 78 3.7 Enzymatic assay of AneH and AaPIP with aculene A 80 3.8 Conclusion 84 4 Reference 87 5 Appendix 91 | |
| dc.language.iso | en | |
| dc.subject | Norsesquiterpenoid | zh_TW |
| dc.subject | aculene | zh_TW |
| dc.subject | 脯氨酸胺?? | zh_TW |
| dc.subject | 生合成 | zh_TW |
| dc.subject | biosynthesis | en |
| dc.subject | Norsesquiterpenoid | en |
| dc.subject | proline iminopeptidase | en |
| dc.subject | aculene | en |
| dc.title | 解析棘孢麴黴之倍半萜類-Aculenes的生合成途徑 | zh_TW |
| dc.title | Elucidation and Characterization of Aculenes Norsesquiterpenes Biosynthesis from Aspergillus aculeatus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳世雄(Shih-Hsiung Wu),梁博煌(Po-Huang Liang),陳榮傑(Rong-Jie Chein) | |
| dc.subject.keyword | Norsesquiterpenoid,aculene,脯氨酸胺??,生合成, | zh_TW |
| dc.subject.keyword | Norsesquiterpenoid,aculene,proline iminopeptidase,biosynthesis, | en |
| dc.relation.page | 185 | |
| dc.identifier.doi | 10.6342/NTU201801916 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-07-26 | |
| 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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