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Nostoc sp. PCC 7120의 카로티노이드 절단 산소 첨가 효소의 대장균으로의 이종 발현 및 특성 연구

Heterologous expression and characterization of a carotenoid cleavage oxygenase of Nostoc sp. PCC 7120

초록/요약

Carotenoid cleavage dioxygenases (CCDs) produce biologically active apocarotenoids. Their biosynthesis is initiated by the oxidative cleavage of double bonds in a carotenoid backbone, leading to aldehydes and/or ketones in each product at the site of cleavage. Apocarotenoids of higher organisms is well known. On the other hand, very little is known about apocarotenoids and CCDs in microorganisms. This study surveyed activities of carotenoid cleavage dioxygenases in Nostoc sp. PCC 7120, encoded by the ORF (open reading frame) all4284, all4895, all1106 here named Nos1, Nos2, Nos3, respectively. Confirmed three gene expression. Also to determine the biochemical and enzymological characterization of Nos2, it was expressed in Escherichia coli and purified for in vitro assays. The highest activity was obtained under conditions of pH 7.0 at 37 ºC with 0.5 mM FeSO4 using β-apo-8’-carotenal as a substrate. The most effective detergent was Tween 40 compare to Tween 20, Tween 80, Triton-X100 and β-octylglucoside. Under optimum conditions, the kinetic values of Nos2 for the β-apo-8’-carotenal were Km = 28 μM and Vmax = 0.79 nmol-1 mg-1 min-1. Surprisingly, unlike previous reports, Nos2 cleaves the central 15,15’ double bond and/or an eccentric double bond at the 13,14(13’,14’) double bond of C30 β-apo-8’-carotenal, leading to C20 retinal, C22 β-apo-14’-carotenal and C18 β-apo-13-carotenal. Moreover, Nos2 could not use β-carotene as a substrate. To investigate the substrate specificity, Nos2 gene was co-expressed with C30 or C40 carotenoid biosynthetic genes in E. coli JM109. When co-expressed with 4,4’-diaponeurosporene(C30) pathway, C17 aldehyde was mainly produced as a result of cleavage at fifth double bond from the fully conjugated end of backbone. In both cases of 4,4’-diaponeurosporenal(C30) and 4,4’-diaponeurosporen-4-oic acid(C30), fourth double bond was cleaved by Nos2 and C22 aldehyde was detected. Also Diapotorulene (C30) cleaved into C17 aldehyde included β-ionone ring. Diapotorulenal (C30) cleaved into C22 aldehyde included β-ionone ring. The experimental results diapo- β-carotene(C30), lycopene(C40), β-carotene(C40), zeaxanthin(C40) did not cleaved by the Nos2. But, when co-expressed with torulene(C40) pathway, β-apo-8’-carotenal is produced and used as the substrate of Nos2. Finally, retinal was mainly produced from the β-apo-8’-carotenal. Directed evolution of Nos2 was performed and visually screening method (schiff’s method) was developed for diversification of substrate specificity. About 5,000 mutant clones, only 3 clones were confirmed that to use β-carotene as a substrate in vitro.

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목차

List of Tables
List of Figures
Abstract
I. Introduction
II. Materials and Methods
1. Gene cloning
2. Culture condition and Strain
3. Protein Expression and Purification
4. Enzyme kinetic assays
5. Kinetic analyses Effects of pH and Temperature on Enzyme activity
6. In vivo analysis of carotenoid cleavage activity
7. Substrate specificity
8. Analytical methods
8.1. LC and LC/MS analysis
8.2. GC-MS analysis
8.3. Kinetic analysis
9. Directed evolution
9.1. Random mutagenesis and recombination by Error-prone PCR
9.2 Saturation mutagenesis
9.3. High throughput screening (HTS) : Schiff’s method
III. Results and Discussion
1. Gene cloning and expression of soluble carotenoid cleavage dioxygenases
2. Protein purification
3. In vitro carotenoid cleavage by Nos2 enzymes
4. Determination of the retinal product of the Nos2 protein
5. Effects of pH and Temperature on the Activity of the Nos2 protein
6. Kinetics analyses of the Nos2 protein
7. Co-expression of Nostoc Dioxygenases in Carotenoid accumulating E. coli
8. Substrate specificity
9. Site-Saturation mutagenesis of Nos2
VI. Conclusion
V. References
Abstract in Korean
Acknowledgements

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