Title Page
Contents
ABSTRACT 10
Ⅰ. Introduction 12
Ⅱ. Materials and Methods 14
2.1. Bacterial strains and culture conditions 14
2.2. Genome mining of the target cluster by using EFI tool, phylogenetic tree analysis, antiSMASH tool 14
2.3. Fosmid library construction & Screening 15
2.4. Gene cluster cloning 16
2.5. CRISPR/Cas9-based genome editing 16
2.6. Heterologous expression of nucleoside gene cluster in Streptomyces albus J1074 17
2.7. Nucleoside compounds production and isolation 18
2.8. Elucidating of chemical structure, LC/MS analysis for biosynthetic pathway and bioactivity assay 18
Ⅲ. Results and Discussion 20
3.1. Genome mining and cloning of nucleoside gene cluster 20
3.1.1. Gene discovery by using EFI tool 20
3.1.2. Fosmid library construction and screening 21
3.1.3. Assembly of fosmids via TAR cloning 22
3.2. Heterologous expression and determining nucleoside gene cluster boundary 25
3.2.1. Heterologous expression of cluster 3 in S. albus 25
3.2.2. Determining the nucleoside gene cluster boundary 25
3.3. Determination of the nucleoside compound structure 29
3.4. Revealing the biosynthetic pathway of nucleoside compounds 44
3.4.1. CRISPR/Cas9 based genome knock out experiment 44
3.4.2. Core gene expression study 44
3.4.3. Biosynthetic pathway of nucleoside compound 49
3.5. Bioactivity assay 52
Ⅳ. Conclusion 53
References 55
ABSTRACT (in Korean) 58
Table 1. List of primers used in this study. 23
Table 2. Gene information table of nucleoside gene cluster 27
Table 3-1. Compound 1 structure and NMR table 40
Table 3-2. Compound 2 structure and NMR table 41
Table 3-3. Compound 3 structure and NMR table 42
Table 3-4. Compound 5 structure and NMR table 43
Table 4. The results of IC₅₀ test of the metabolites. (256 μg/mL) 52
Figure 1. The biosynthetic pathway of preQ₀. GTP is converted to preQ₀ in four enzymatic step through FolE, QueD, QueE, QueC, as shown.... 13
Figure 2. The phylogenetic Tree of QueC gene. Total 51 unique sequences of queC gene was aligned by MUSCLE method and analyzed by Maximum 21
Figure 3. a. The schematic diagram depicts the cloning of the predicted nucleoside gene cluster (24kb) along with flanking genes (8kb) on both... 24
Figure 4. The results presented in Figure 4 depict the analysis conducted via HPLC in S. albus. after conjugating each of the re-captured pTARb... 26
Figure 5A. ¹H NMR spectrum (DMSO-d₆ 600 MHz) of compound 1 29
Figure 5B. ¹³C NMR spectrum (DMSO-d₆ 150 MHz) of compound 1 30
Figure 5C. COSY NMR spectrum (DMSO-d₆ 600 MHz) of compound 1 30
Figure 5D. HSQC NMR spectrum (DMSO-d₆ 600 MHz) of compound 1 31
Figure 5E. HMBC NMR spectrum (DMSO-d₆ 600 MHz) of compound 1 31
Figure 5F. ¹H NMR spectrum (DMSO-d₆ 600 MHz) of compound 2 32
Figure 5G. ¹³C NMR spectrum (DMSO-d₆ 150 MHz) of compound 2 32
Figure 5H. COSY NMR spectrum (DMSO-d₆ 600 MHz) of compound 2 33
Figure 5I. HSQC NMR spectrum (DMSO-d₆ 600 MHz) of compound 2 33
Figure 5J. HMBC NMR spectrum (DMSO-d₆ 600 MHz) of compound 2 34
Figure 5K. ¹H NMR spectrum (DMSO-d₆ 600 MHz) of compound 3 34
Figure 5L. ¹³C NMR spectrum (DMSO-d₆ 150 MHz) of compound 3 35
Figure 5M. COSY NMR spectrum (DMSO-d₆ 600 MHz) of compound 3 35
Figure 5N. HSQC NMR spectrum (DMSO-d₆ 600 MHz) of compound 3 36
Figure 5O. HMBC NMR spectrum (DMSO-d₆ 600 MHz) of compound 3 36
Figure 5P. ¹H NMR spectrum (DMSO-d₆ 600 MHz) of compound 5 37
Figure 5Q. ¹³C NMR spectrum (DMSO-d₆ 150 MHz) of compound 5 37
Figure 5R. COSY NMR spectrum (DMSO-d₆ 600 MHz) of compound 5 38
Figure 5S. HSQC NMR spectrum (DMSO-d₆ 600 MHz) of compound 5 38
Figure 5T. HMBC NMR spectrum (DMSO-d₆ 600 MHz) of compound 5 39
Figure 6. The figure presents a comparison of HPLC metabolite analyses after cloning pTARb vectors with gene deletions achieved through... 46
Figure 7. Since the known function of ORF12(QueC) is the conversion of 7-deaza-7-carboxyguanine(CDG) to PreQ₀ which contains the nitrile group.... 47
Figure 8. The results from LC/MS analysis of the two peaks observed in the core gene expression construct are presented. It was confirmed that... 48
Figure 9. LC/MS data of S. albus_NUC03_△ORF 8. 50
Figure 10. Biosynthetic pathway of nucleoside compound. 51