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재조합 이콜라이에서 c30카로티노이드 생합성회로 및 메발로네이트 생합성회로 구축

Construction of novel C30 carotenoid and mevalonate pathway in recombinant Escherichia coli

초록/요약

Global warming and depletion of fossil fuels are driving research on the production of compounds and biofuels using microbial cell factories rather than chemical synthesis. In previous studies, to obtain high-efficiency foreign genes from genetic information they were searched in a database, obtained by degenerating PCR using degenerate primer or by using a fosmid library construction. However, these old methods are costly and often inaccurate. Recent development of next generation sequencing can be used to quickly and easily obtain information on high-efficiency genes. Metabolic engineering is implemented for developing microbial cell factories using genetic information from next generation sequencing. Construction of heterologous biosynthesis pathways is possible by using genetic information. Many microorganisms such as E. coli or yeast can be used to produce valuable materials on an industrial scale in an efficient manner. A noble type of strain, Planococcus faecalis AJ003T, isolated from the feces of the Antarctic penguin synthesizes a rare C30 carotenoid, glycosyl-4,4'-diapoinurosporphorin-4'-ol-4-ol acid. The genome of P. faecalis AJ003T comprises a 3,495,892bp circular chromosome (40.9 % G+C content), and is devoid of any extrachromosomal plasmids. Genome annotation analysis revealed that it six genes involved in the carotenoid pathway. The function and complementation of 4,4'-diapophytoene synthase (CrtM), 4,4'-diapophytoene desaturase (CrtN), 4,4-diaponeurosporene oxidase (CrtP) and aldehyde dehydrogenase (aldH) were analyzed in E. coli. Complementation of each gene of P. faecalis AJ003T with carotenoid biosynthetic module of Staphylococcus aureus in E. coli assigned the function of each gene. As a result, 4,4′-diaponeurosporenoic acid, 4,4′-diapolycopene-4,4’-dioic acid, 4,4′-diapolycopen-4′-al-4-oic acid and 4,4′-diapolycopenoic acid were detected by HPLC analysis. In conclusion, by using NGS, the C30 carotenoid gene was efficiently obtained and microbial cell factories were constructed by the metabolic engineering technique to produce 4,4'-diapolycopene-4,4'-dioic acid and 4,4'-diaponeurosporenoic acid metabolic pathway. A novel species, Flavobacterium kingsejong WV39T, isolated from the feces of the Antarctic penguin overproduces zeaxanthin as a main carotenoid. The complete genome of F. kingsejong WV39T is made up of a single circular chromosome (4,224,053 bp, 39.8% G +C content). Genome annotation analysis revealed that it has a five zeaxanthin genes and six mevalonate pathway genes that produce the IPP, a precursor of zeaxanthin. Functional analysis of the mevalonate enzyme from F. kingsejong WV39T in E. coli was completed. Each gene of F. kingsejong WV39T was complemented with the mevalonate biosynthesis module of Enterococcus faecalis and Flavobacterium faecalis WV33 in E. coli. As a result, the titer, yield and productivity of mevalonate were 64.02 ± 1.43 g/L, 0.24 ± 0 g/g and 1.06 ± 0 g/L/h, respectively, through fermentation using the plasmid system and pH stat method. To develop industrial strains that overcome the instability of plasmids, the 5' untranslated region (5' UTR) of mRNA was introduced for the genome editing, mRNA expression and stability of E coli. The mevalonate titer, yield and productivity of the strain was 14.07 ± 0.12 g/L, 0.21 ± 0.01 g/g and 0.46 ± 0.01 g/L/ h, respectively. The yield increased by about 7 folds as compared with that of the strain editing of mevalonate gene. In conclusion, the mevalonate pathway gene found in NGS was introduced into E. coli and the possibility of producing mevalonate was showed by flask culture and fed batch. In conclusion, we succeeded in securing high efficiency genes easily and quickly by using the next generation sequencing and confirmed the function of genes by introducing them into E. coli. These results using engineering tools will provide information for further application and engineering of carotenoids and mevalonate.

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목차

ABSTRACT i
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS x
Chapter 1. General Introduction - 1 -
1.1 Metabolic engineering of microoranisms - 2 -
1.2 Exploration of a genome of novel microorganisms - 3 -
1.3 Microbial production of Carotenoids and Mevalonate - 6 -
1.4 MEP pathway - 9 -
1.5 MVA pathway - 10 -
1.6 Aims of this study - 12 -
Chapter 2. The complete genome sequences of Planococcus faecalis AJ003T for construction of C30 carotenoid and Flavobacterium kingsejong WV39T for mevalonate production - 13 -
2.1 Abstract - 14 -
2.2 Introduction - 15 -
2.3 Materials and methods - 17 -
2.4 Result and discussion - 18 -
2.4.1 Completed of genome sequencing of P. faecalis AJ003T - 18 -
2.4.2 Completed of genome sequencing of F. kingsejong WV39T - 23 -
2.5. Conclusion - 28 -
Chapter 3. Construction of novel C30 carotenoid biosynthesis pathway of Planococcus faecalis AJ003T in Escherichia coli - 29 -
3.1 Abstract - 30 -
3.2 Introduction - 31 -
3.3 Materials and methods - 33 -
3.3.1 Sequencing of genome - 33 -
3.3.2 Bacterial strains, plasmids, and culture contidion - 33 -
3.3.3 Cloning and Constrcution of synthetic expression module - 33 -
3.3.4 Culture condition - 34 -
3.3.5 Isolation of carotenoids - 34 -
3.3.6Analysis of carotenoids - 35 -
3.4 Result - 39 -
3.4.1 Identification of C30 carotenoid gene - 39 -
3.4.2 Functional analysis of noble C30 carotenoid biosynthesis cluster in E.coli - 42 -
3.4.3 Identification of crtP and aldH - 46 -
3.5 Conclusion - 52 -
Chapter 4. Construction of mevalonate biosynthesis pathways of Flavobacterium kingsejong WV39T for production of mevalonate in Escherichia coli - 53 -
4.1 Abstract - 54 -
4.2. Introduction - 55 -
4.3. Materials and methods - 57 -
4.3.1. Bacterial strains, plasmids and growth conditions - 57 -
4.3.2. Cloning and synthetic module construction - 57 -
4.3.3. Protein expression - 58 -
4.3.4. Media and culture condition - 58 -
4.3.5 Fermentation conditions - 58 -
4.3.6 Analytical methods - 59 -
4.3.7 Genome Engineering - 59 -
4.3.8 Quantitative Real-Time PCR - 60 -
4.4. Result and discussion - 69 -
4.4.1 Identification of HMG-CoA reductase and HMG-CoA synthase - 69 -
4.4.2. Construction of synthetic expression modules - 71 -
4.4.3 Fermentation in bioreactor - 72 -
4.4.4 Construction of genome edited strain - 76 -
4.4.5 Engineering of Genome Editing Strain - 80 -
4.4.6 Improvement of the production yield by UTR insertion - 82 -
4.5 Conclusion - 85 -
5. CONCLUSION - 86 -
REFFERENCE - 88 -
ABSTRACT IN KOREAN - 98 -

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