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Graduate School of Ajou University Department of Molecular Science and Technology 3. Theses(Master)

Redesign of biosynthesis pathway of carotenoids and production of structurally novel carotenoids in heterologous host, Escherichia coli

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dc.contributor.advisor이평천-
dc.contributor.authorKim, Se Hyeuk-
dc.date.accessioned2018-11-08T08:21:19Z-
dc.date.available2018-11-08T08:21:19Z-
dc.date.issued2014-02-
dc.identifier.other16163-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/12981-
dc.description학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2014. 2-
dc.description.tableofcontentsCONTENTS LIST OF TABLES…………...……………………...........................…..….……...i LIST OF FIGURES……………………...……..…………………….……………ii ABSTRACT…………………………………………………………………..……iv Chapter 1. General Introduction………………………………………………..1 1.1 Introduction…………………….……………………..…………………...2 1.2 MEP pathway………………………………………………..………………5 1.3 MEV pathway……………………….…………………….…………………6 1.4 Microbial production of carotenoids………………………………….……..8 1.5 Aims of This study ………………………………….………………………9 Chapter 2. Astaxanthin dirhamnoside biosynthesis pathway in Sphingomonas lacus PB304T…………………………………………..………………………….12 2.1 Abstract……………………………………………………….……………13 2.2 Introduction………………..…………………..…………………………14 2.3 Materials and Methods……………………………………………………..17 2.3.1. Bacterial strains, plasmids, and growth conditions……………..….17 2.3.2. Phylogenetic analysis of 16S rRNA………………………………….17 2.3.3. Recombinant DNA techniques…………………………….…………18 2.3.4. Fosmid library construction…………………………….……………18 2.3.5. Colony hybridization………………………………….……………..19 2.3.6. Gene cloning…………………………………………………………20 2.3.7. Carotenoid isolation and structural analysis………………..………20 2.4 Results…………………………………………………………………..…24 2.4.1. Taxonomic analysis of the isolated strain PB304……………….……24 2.4.2. Carotenoid profile of Sphingomonas lacus PB304T and structural assignment of the major Sphingomonas lacus PB304T carotenoid…24 2.4.3. Identification of CrtZ, CrtB, CrtI, and CrtY…………………………28 2.4.4. Identification of CrtW and CrtX…………………………………….30 2.4.5. Functional analysis of the putative carotenogenic enzymes from Sphingomonas lacus PB304T………………………………………32 2.4.6. Phylogenetic analysis of Sphingomonas lacus PB304T CrtX………...35 2.5. Discussion……………………………………………………..…………37 Chapter 3. Functional expression and extension of the staphylococcal staphyloxanthin biosynthetic pathway in Escherichia coli…..40 3.1. Abstract…………………………………………..…………………….…41 3.2. Introduction…………….………………………………..………………42 3.3. Materials and Methods…………………………………………………….46 3.3.1. Cloning and synthetic module construction……………….…………46 3.3.2. Culture growth for carotenoid production……………………………47 3.3.3. Isolation of carotenoids………………………………………………47 3.3.4. Preparation of carotenoids for LC/MS……………..………………48 3.3.5. Allelic replacement…………………………………………………48 3.3.6. Analysis of carotenoids………………………………………………49 3.4. Results …………………………………………………………………….52 3.4.1. Reconstruction of a partial staphyloxanthin biosynthetic pathway in E. coli………………………………………………………………….52 3.4.2. Identification of the sixth enzyme in the staphyloxanthin biosynthetic pathway of S. aureus…………………………………………………55 3.4.3. Identification of an enzyme catalyzing the oxidation of carotenoid aldehyde to carboxylic acid in S. carnosus…………………………60 3.4.4. Functional verification of 4,4′-diaponeurosporen-aldehyde dehydrogenase (AldH) in S. aureus using allelic replacement.……61 3.4.5. Expression of the complete staphyloxanthin pathway in E. coli and characterization of staphyloxanthin-like compounds produced in engineered E. coli……………………………………………………65 3.5. Discussion………………………………………………………………71 Chapter 4. Investigation of biological activity of natural and non-natural C30 carotenoids ………………...……………………………….……….73 4.1. Abstract…………………………………………………………………….74 4.2. Introduction……………………………………………………..………75 4.3. Materials and Methods……………………………………………………79 4.3.1. Bacterial strains, plasmids, and growth conditions………………….79 4.3.2. Gene cloning and construction of C30 carotenogenic gene module…79 4.3.3. Isolation of carotenoids………………………………………………80 4.3.4. Analysis of carotenoids………………………………………………80 4.3.5. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenge assay……81 4.3.7. Cytotoxicity tests and neurogenesis of rBMSCs……………………82 4.4. Results……………………………………………………………………85 4.4.1. Manipulation of C30 acyclic carotenoid biosynthesis pathway………85 4.4.2. Manipulation of C30 monocyclic carotenoid biosynthesis……………87 4.4.3. Manipulation of bicyclic C30 carotenoids pathway………………….90 4.4.4. Radical scavenging activity measurement of various structures of C30 carotenoids……………………….…………………………………..93 4.4.5. Neuronal differentiation of rat Bone Marrow Mesenchymal stem cells (rBMSCs) induced by 4,4’-diapotorulene……………………………96 4.5. Discussion…………………………………………………………………99 Chapter 5. Proposed anticancer mechanisms of two major saffron carotenoids, crocin and crocetin, on cancer cell lines…………………………102 5.1. Abstract…………………………………………………………………103 5.2. Introduction………………………………………………………………104 5.3. Materials and Methods…………………………………………………106 5.3.1. Reagents……………………………………………………………106 5.3.2. Cell culture……………………………………………………….…106 5.3.3. Cell viability - MTT assay…………………………………………106 5.3.4. Western blot analysis………………………………………………107 5.3.5. Reverse transcriptase PCR (RT-PCR)…………………………...…108 5.3.6. Reporter assay………………………………………………………108 5.3.7. Detection of ROS………………………………………..…………109 5.3.8. LDH activity assay…………………………………………………109 5.3.9. Statistical analysis…………………………………………………110 5.4. Results……………………………………………………………….….111 5.4.1. Cytotoxicity of crocin and crocetin on 5 human cancer cell lines…111 5.4.2. Intracellular ROS levels in crocin/crocetin-treated HeLa cells……114 5.4.3. ROS-associated cytotoxicity of crocin/crocetin…………………….114 5.4.4. Crocin/crocetin-mediated Nrf2 activation…………………………118 5.4.5. Inhibitory activity of crocin and crocetin on the expression of LDHA in HeLa cells…………………….……………………………………120 5.5. Discussion……………………………………………………………....122 Chapter 6. Simultaneous transcriptional and translational regulation for tuning protein expression level in Escherichia coli ……………126 6.1. Abstract…………………………………………………………………127 6.2. Introduction…………………………………………………………….128 6.3. Materials and Methods…………………………………………………130 6.3.1. Bacterial strains, plasmids, and growth conditions ………………130 6.3.2. Recombinant DNA techniques …………………………………....130 6.3.3. Construction of reporter plasmid …………………………………..130 6.3.4. β-Galactosidase assay …………………………………….………131 6.4. Results…………………………………….……………………………..134 6.4.1. Construction of synthetic expression cassette (SEC) library...…….134 6.4.2. Screening of SEC library with β-galactosidase assay ……………137 6.4.3. Sequence analysis of the SEC library.…..…………………………140 6.5. Discussion………………………………………………………………142 7. Conclusion……………….…………………………………………………144 8. References……………………...……………….……………………………146 ABSTRACT IN KOREAN………………………………..…….…………….…169 ACKNOWLEDGEMENTS………………..………………….…………....……172-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.titleRedesign of biosynthesis pathway of carotenoids and production of structurally novel carotenoids in heterologous host, Escherichia coli-
dc.title.alternativeSe Hyeuk Kim-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameSe Hyeuk Kim-
dc.contributor.department일반대학원 분자과학기술학과-
dc.date.awarded2014. 2-
dc.description.degreeMaster-
dc.identifier.localId609676-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000016163-
dc.subject.keywordcarotenoid-
dc.subject.keywordheterologous expression-
dc.subject.keywordE. coli-
dc.description.alternativeAbstractIncreasing demand on the development of sustainable processes is raising importance of the microbial production of natural products, chemicals, and fuels. This trend has driven recent research on the production of isoprenoids such as terpenoids and carotenoids in heterologous hosts. In particular diverse structures of carotenoids, natural isoprenoid pigments, were produced in engineered heterologous microorganisms due to their beneficial functions to humans. As an attempt to engineer diverse structures of carotenoids of biological significances, 1) biochemical and genetic elucidation of biosynthesis pathways of carotenoids, 2) heterologous production of various novel structures of C30 carotenoids, 3) mechanism studies on biological activities of carotenoids, and 4) regulatory engineering for fine-controlling pathway enzyme expression have been carried out. First, the biosynthesis pathway of astaxanthin dirhamnoside of novel species, Sphingomonas lacus PB304T, was elucidated genetically and biochemically. Two gene clusters, crtZBIY and crtWX, encoding six astaxanthin dirhamnoside biosynthesis enzymes were discovered from a fosmid library, and six genes were annotated by blast analysis. Complementation of each gene of S. lacus PB304T with carotenogenic gene modules of Pantoea agglomerans in E. coli assigned the function of each gene except for rhamnosyltransferase, CrtX. Second, a missing enzyme, 4,4’-diaponeurosporen-aldehyde dehydrogenase (AldH), that is involved in the biosynthesis of staphyloxanthin in Staphylococcus aureus was identified. Through heterologous expression of biosynthesis pathway of staphyloxanthin in E. coli and knockout strategy in S. aureus, the new function of AldH was assayed, and AldH was found to be an essential enzyme in the biosynthesis of staphyloxanthin. Heterologous expression of complete pathway enzymes of staphyloxanthin in E. coli resulted in biosynthesis of a few staphyloxanthin-like compounds, which have altered fatty acid acyl chains, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes in a heterologous host E. coli. Third, nine novel structures of C30 carotenoids using in vitro evolved 4,4’-diapophytoene desaturase (CrtN) and lycopene cyclase (CrtY) was created by extending 4,4’-diaponeurosporene, 4,4’-diapotorulene, and 4,4’-diapo-β-carotene pathway enzymes in E. coli. Two acyclic, three monocyclic, and four bicyclic C30 carotenoids were produced. 4,4’-diapolycopen-dial exhibited the highest radical scavenging activity (4.4 fold compare with DL-α-tocopherol), and 4,4’-diapotorulene induced neuronal cells-like morphological changes to rat bone marrow mesenchymal stem cells (rBMSCs), demonstrating potential uses of novel C30 carotenoids. Fourth, anticancer activities of two major glycosylated (crocin) and carboxylic (crocetin) carotenoids were investigated on 5 human cancer cell lines, and their possible anticancer mechanisms were proposed. Crocetin showed a 7-fold higher cytotoxicity than crocin, suggesting that structural differences account for the different efficacies between them. It was further proved by higher induction of intracellular ROS in HeLa cells monitored by FACS analysis. Induction of nuclear factor E2-related factor 2 (Nrf2) was also monitored, and inhibitory effect of crocin and crocetin on lactate dehydrogenase A (LDHA), one of the targets for chemoprevention in cancer cells, was measured. Crocetin showed higher effect on induction of Nrf2 and inhibition of LDHA, indicating crocetin and crocin have different mechanisms for the observed cytotoxicity in cancer cell lines. Finally, synthetic expression cassettes (SEC) were constructed for fine-tuning pathway enzyme expression. Sequences of promoter and ribosomal binding regions were simultaneously randomized and screened based on the β-galactosidase activity. The strength of SEC was adjusted between 5 to 8500 Miller Unit (MU) based on the activity level. However, no significant features were found from sequence analysis and in silico calculation of translation initiation rate. In conclusion, through systematic studies on heterologous biosynthesis of diverse structures of carotenoids by using synthetic expression modules and combinatorial biosynthesis, 2 new biosynthetic pathways were identified and characterized genetically and biochemically, over 10 novel structures of carotenoids were created, and their biological activities and cellular mechanisms were investigated. These results with SECs and other engineering tools will be served invaluable information for further application and engineering of isoprenoids including carotenoids.-
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