Title Page
Abstract
Contents
Abbreviation 20
CHAPTER Ⅰ. General introduction 23
1.1. Nocardia spp. and its bioactive compounds 24
1.2. Genome analysis of biosynthetic pathway of IBR-3 in Nocardia sp. CS682 25
1.3. Study of gene functions of IBR-3 gene cluster in Nocardia sp CS682. 28
1.3.1. Polyketide synthase 28
1.3.2. Methyltransferase 31
1.4. Metabolic engineering of microbes and biotransformation 32
1.5. Objectives of this study 33
CHAPTER Ⅱ. Materials and methods 35
2.1. General procedure 36
2.1.1. Reagents and enzymes 36
2.1.2. Bacterial strains, vectors, culture conditions, and recombinant plasmids 36
2.1.3. Preparation of competent cell and transformation 39
2.2. Culture parameters 40
2.2.1. Culture media 40
2.2.2. Culture conditions 40
2.3. DNA manipulation and sequence analyses 41
2.3.1. Polymerase chain reaction (PCR) 41
2.3.2. Purification and cloning of PCR product 43
2.3.3. Preparation of competent cell and transformation of plasmid in E. coli 44
2.4. Expression, purification, and quantification of protein 45
2.4.1. Cloning, expression, and characterization of ThnA, ThnM1, and ThnM3 45
2.4.2. General properties of enzymes and their enzyme kinetics determination 46
2.5. Flexibility of Acceptor substrates by enzymes 48
2.6. Generation of recombinant strains 48
2.6.1. Transformation of Nocardia sp. CS682 50
2.6.2. Transformation of S. lividans TK24 51
2.7. Whole-cell biotransformation 51
2.8. Analytical method 52
2.8.1. HPLC 52
2.8.2. Preparative HPLC 52
2.8.3. Mass and NMR analyses 53
2.9. Biological activity tests 53
2.9.1. Antimicrobial activities 53
2.9.2. Anticancer activity tests 55
CHAPTER Ⅲ. Functional characterization of a regiospecific sugar-O-methyltransferase, ThnM1 56
3.1. Background 57
3.2. Results and Discussion 59
3.2.1. Sequence and phylogenetic analysis of ThnM1 59
3.2.2. Cloning, expression, and purification of ThnM1 62
3.2.3. Substrate promiscuity of ThnM1 63
3.2.4. Effects of incubation temperature, pH, and cofactors on enzyme activity 66
3.2.5. Whole-cell biotransformation of quinizarin 69
3.2.6. Analysis and characterization of a methylated derivative of quinizarin 70
3.2.7. Cytotoxicity test 75
CHAPTER Ⅳ. Microbial Biosynthesis of chrysazin-8-O-α-L-2'-O-methylrhamnoside by using ThnM1 76
4.1. Background 77
4.2. Results and Discussion 79
4.2.1. Biosynthesis of CR and CRM 79
4.2.2. Purification and Structural Elucidation of the Metabolite 82
4.2.3. Anticancer Activities 87
4.2.4. Antimicrobial Bacterial Activities 89
CHAPTER Ⅴ. Functional characterization of regioselective naphthalene O-methyltransferase from Nocardia sp. CS682 93
5.1. Background 94
5.2. Results and Discussion 95
5.2.1. Sequence and phylogenetic analysis of ThnM3 95
5.2.2. Cloning, expression, and purification of ThnM3 97
5.2.3. Substrate promiscuity and in vitro assays for the O-methyltransferase activity of ThnM3 98
5.2.4. Effects of cofactors, pH, and temperature on enzyme activity 102
5.5. Biosynthesis of 1,8-dimethoxynapthalene 103
5.6. Purification and structural elucidation of the metabolite 104
CHAPTER Ⅵ. Overexpression of SARP regulator for production of furanonaphthoquinone in Nocardia sp. CS682 108
6.1. Background 109
6.2. Results and Discussion 111
6.2.1. Overexpression of SARP regulator genes 111
6.2.2. Fermentation, Isolation, and Structural elucidation of NOC-IBR1 and NOC-IBR2 113
6.2.3. Bioinformatic analysis of the genome of Nocardia sp. CS682 and annotation of the ifn gene cluster 117
6.2.4. Identification of the fnq Cluster and a proposed biosynthetic pathway for FNQs in Nocardia 119
6.3. Biological Activities 121
6.3.1. Anticancer activities 121
6.3.2. Antimicrobial bacterial activities 123
CHAPTER Ⅶ. Identification of a Type III Polyketide Synthase (1,3,6,8-Tetrahydroxynaphthalene Synthase) from Nocardia sp. CS682. 128
7.1. Background 129
7.2. Results and Discussion 131
7.2.1. Sequence and phylogenetic analysis of ThnA 131
7.2.2. In vivo heterologous expression of ThnA and analysis 134
7.2.3. RNA Isolation and Real-Time PCR analysis 136
CHAPTER Ⅷ. Overall conclusion and future perspectives 137
References 141
국문초록 150
Appendix 153
Conferences 154
Table 1.1. Different compound predicted from Nocardia sp. CS682DR based on MASS analysis. 26
Table 1.2. Putative genes involved in the biosynthetic gene cluster of compound IBR-3. 27
Table 2.1. List of strains, vectors, and plasmids used in this study. 38
Table 2.2. List of primers and DNA sequences used in this study. 43
Table 3.1. The Kinetic parameter of ThnM1 with QR and SAM at different substrate concentrations. 67
Table 3.2. Effects of SAM concentration in the mutant strain (E. coli-speD, E. coli-MetK, E. coli-Metk/SpeD, E. coli-S2) compared with the wild-type strain. 70
Table 3.3. Comparison of ¹H- and ¹³C-NMR chemical shifts of quinizarin, quinizarin-4-O-α-L-rhamnoside, and quinizarin-4-O-α-L-2'-O-methylrhamnoside measured in DMSO-d6 solvent. 73
Table 3.4. Cell cytotoxicity assay of quinizarin and quinizarin-4-O-α-L-2'-O-methylrhamnoside against different cancer cell lines with IC₅₀(µM) values. 75
Table 4.1. Comparison of ¹H- and ¹³C-NMR chemical shifts of chrysazin, CR, and CRM measured in DMSO-d6 solvent 84
Table 4.2. IC₅₀ values of three compounds against AGS, Huh7, and HL60 cell lines 88
Table 4.3. MIC values of compound chrysazin, CR, CRM, and erythromycin (Erm) against 9 strains. 92
Table 4.4. MBC values of compound chrysazin, CR, CRM, and erythromycin (Erm) against 9 strains 92
Table 5.1. Comparison of ¹H- and ¹³C-NMR chemical shifts of 1,8 dihydroxy naphthalene, and 1,8-dimethoxy naphthalene measured in DMSO-d₆ solvent. 107
Table 6.1. Comparison of ¹H- and ¹³C-NMR chemical shifts of NOC-IBR1, and NOC-IBR2 measured in DMSO-d6 solvent. 114
Table 6.2. Predicted functions of genes in the fnq gene cluster. 120
Table 6.3. Anticancer (IC50 (μM)) potential of NOC-IBR1 and NOC-IBR2 against different cell lines. 122
Table 6.4. Antibacterial activities test against Gram-positive and Gram-negative bacteria via disk diffusion. The zone of inhibition (diameter) due to NOC-IBR1, NOC-IBR2, erythromycin,... 124
Table 6.5. MIC values of compound NOC-IBR1, NOC-IBR2, and erythromycin (Erm) against 9 strains. 125
Table 6.6. MBC values of compound NOC-IBR1, NOC-IBR2, and erythromycin (Erm) against 9 strains 127
Figure 1.1. Different bioactive compounds produced from various Nocardia spp. 24
Figure 1.2. Ultra-pressure liquid chromatography (UPLC) chromatogram of extract from Nocardia sp.CS682 and Nocardia sp. CS682DR. 25
Figure 1.3. The putative biosynthetic gene cluster and proposed biosynthetic mechanism of compound IBR-3. 27
Figure 1.4. Representative structure of PKS enzymes. (A) Iterative type I PKS, (B) non-iterative, modular type I PKS, (C) minimal type II PKS, and (D) type III PKS. Abbreviations: KS... 29
Figure 1.5. Chemical structures of major bacterial type III polyketide synthase (PKS) products. 31
Figure 1.6. SAM-dependent methyltransferases (MTs) reaction of different substrates 32
Figure 3.1. The putative biosynthetic gene cluster and proposed biosynthetic pathway of IBR-3. Compound 1: 3,6,8-trimethoxy naphthalen-1-ol; 2: 1-(α-L-6-deoxy- mannopyranosyloxy)-... 60
Figure 3.2. Evolutionary relationship of methyl transferase family proteins from different sources. The name of the enzyme, strain and accession number of the amino acid sequence is... 61
Figure 3.3. Homologous amino acid sequences alignment of ThnM1 with selected previously characterized methyltransferase. Sequences alignment of ThnM1 with other O-... 62
Figure 3.4. 12% SDS-PAGE analysis of heterologously overexpressed ThnM1 protein in E. coli BL21 (DE3). Lane 1: clear lysate of ThnM1; Lane 2: unclear lysate of ThnM1; Lane 3: Insoluble... 63
Figure 3.5. A. Structures of different substrates that are accepted by ThnM1in in vitro reaction. B. Reaction scheme of methylation of quinizarin-4-O-α-L-rhamnoside (QR) by ThnM1 in... 66
Figure 3.6. Probing of ThnM1 assay conditions and determination of conditions for measuring initial velocity. A) Effect of different temperatures on the activity of purified ThnM1. B) Effect... 68
Figure 3.7. Enzyme-Kinetics characteristics of ThnM1. Kinetic analysis using a variable concentration of QR (5-200 µM), ThnM1 (2 µg), SAM (2 mM), MgCl₂ (2 mM), and Tris-HCl... 68
Figure 3.8. Whole-cell biotransformation of quinizarin to QRM by engineered E. coli S2 strain overexpressing anthraquinone glycosyltransferase, sugar-MT (ThnM1), TDP-rhamnose sugar... 71
Figure 3.9. HMBC, COSY, and ROESY correlations of quinizarin-4-O-α-L-2'-O-methylrhamnoside 72
Figure 3.10. 1D NMR spectra of quinizarin-4-O-α-L-2'-O-methylrhamnoside. (a) ¹H NMR spectrum; (b) ¹³C NMR spectrum 74
Figure 3.11. Cell cytotoxicity assay of quinizarin and QRM. Cells were treated with various concentrations (0.0 ~ 200μM) of each compound. 75
Figure 4.1. A scheme showing the pathway of utilizing recombinant Escherichia coli BL21(DE3) for the biosynthesis of chrysazin-8-O-α-L-rhamnoside (CR) and chrysazin-8-O-α-L-2'-O-... 79
Figure 4.2. Whole-cell biotransformation of chrysazin to CR and CRM using engineered E. coli S2 overexpressing anthraquinone glycosyltransferase, sugar-MT (ThnM1), TDP-rhamnose... 81
Figure 4.3. HR-QTOF ESI/MS chromatogram of (A) CRM, (a) UV/VIS of CRM, (B) CR (b) UV/VIS of CR. 82
Figure 4.4. 1D NMR spectra of CR. (a) ¹H NMR spectrum; (b) ¹³C NMR spectrum 85
Figure 4.5. 1D NMR spectra of CRM. (a) ¹H NMR spectrum; (b) ¹³C NMR spectrum 86
Figure 4.6. Cell cytotoxicity assay results of chrysazin, CR, and CRM. 88
Figure 4.7. Antibacterial activity of 1(DMSO), 2(Chrysazin), 3(CR), and 4(CRM) with (i) S. aureus CCARM 0204 (MSSA), (ii) S. aureus CCARM 0205 (MSSA), (iii) S. aureus CCARM... 90
Figure 5.1. The putative biosynthetic gene cluster and proposed biosynthetic pathway of compound 3. Compound 1: 3,6,8-trimethoxy naphthalen-1-ol; 2: 1-(α-L-6-deoxy-... 96
Figure 5.2. Sequence alignment of ThnM3 with other previously characterized methyltransferases. Sequences alignment of ThnM3 with other O-methyltransferases such as... 97
Figure 5.3. SDS-PAGE analysis of heterologously overexpressed ThnM3 protein in E. coli BL21 (DE3). Lane 1: clear lysate of ThnM3; Lane 2: insoluble fraction of ThnM3; Lane 3: unclear... 98
Figure 5.4. A. Structures of different substrates that are accepted by ThnM1in in vitro reactions. B. Reaction scheme of methylation of 1,8-dihydroxy naphthalene by ThnM3 in presence of SAM... 101
Figure 5.5. Probing of ThnM3 assay conditions and determination of conditions for measuring initial velocity. A) Effect of different temperatures on the activity of purified ThnM1. B) Effect... 103
Figure 5.6. Comparison of ¹H-NMR analysis of (A) 1,8-dihydroxy naphthalene and (B) 1,8-dimethoxy naphthalene. 105
Figure 5.7. Comparison of ¹³C-NMR analysis of (A) 1,8-dihydroxy naphthalene and (B) 1,8-dimethoxy naphthalene 106
Figure 5.8. Zoomed-in views of (A) HSQC-DEPT and (B) HMBC analyses of 1,8-dimethoxy naphthalene. The cross peak in HSQC shows the correlation between the proton of the methoxy-... 107
Figure 6.1. HPLC chromatogram of extract from different strains (a) Wild type, Nocardia sp. CS682, (b) PKS deletion mutant Nocardia sp. CS682DR (c) Nocardia sp. CS682DR fnqO... 112
Figure 6.2. 1D NMR spectra of NOC-IBR1. (a) ¹H NMR spectrum; (b) ¹³C NMR spectrum 115
Figure 6.3. 1D NMR spectra of NOC-IBR2. (a) ¹H NMR spectrum; (b) ¹³C NMR spectrum 116
Figure 6.4. A. Reaction scheme of methylation of NOC-IBR1 by ThnM3 in presence of SAM at 40℃ for 2 h (50 mM Tris HCl buffer (pH 8), 2 mM SAM, 2 mM substrate, and 50 µg of... 118
Figure 6.5. Proposed biosynthetic mechanism of compounds NOC-IBR1 and NOC-IBR2. Where FnqE1/E2/E3 = Acyl-carrier protein (ACP), PKS II-KSα, and PKSII-KSβ respectively.... 119
Figure 6.6. Cell cytotoxicity assay of NOC-IBR1 and NOC-IBR2. Cells were treated with various concentrations (0.0 ~ 200μM) of each compound 122
Figure 7.1. Reaction scheme of 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). R = Co-enzyme A (CoA) or the enzyme active site cysteine thiol group 131
Figure 7.2. The putative biosynthetic gene cluster and proposed biosynthetic pathway of compound 3. Compound 1: 3,6,8-trimethoxy naphthalen-1-ol; 2: 1-(α-L-6-deoxy-... 133
Figure 7.3. Sequence alignment of ThnA protein with other known types III PKSs. The comparison was carried out with THNS from Streptomyces coelicolor A3(2) (1U0M), RppA... 133
Figure 7.4. HPLC and LC-ESI/MS analysis of in vivo products. (A) HPLC patterns of the compounds.... 135
Figure 7.5. RT-PCR profile of thnA and RpoB in S. Lividans TK24; Lane 1; RT-PCR of thnA in S. LividansTK24 wild type; Lane 2; RT-PCR of RpoB as a housekeeping gene in S.... 136