Title Page
Contents
ABSTRACT 12
Chapter Ⅰ. General Introduction 16
1.1. Lichens as Sources of Bioactive Secondary Metabolites 17
1.2. The Lichen-Forming Fungi 27
1.3. Biosynthetic Research in Lichen Lichen-forming Fungi 29
1.4. Future Perspectives of Biosynthetic Research in Lichen BGCs 33
Chapter Ⅱ. Evaluation of Antimicrobial Properties of Endolichenic Fungi Against Plant Pathogens 34
1. Introduction 35
2. Materials and Methods 38
2.1. Microbial Strains and Isolates 38
2.2. Mass Production and Extraction of Endolichenic Fungi Crude Extracts 39
2.3. Paper Disk Assay against Phytopathogenic Bacteria 40
2.4. Paper Disk Assay against Phytopathogenic Fungi 41
2.5. HPLC Analysis of the Extracts 41
2.6. LC-MS/MS Analysis of the Extracts 42
3. Results 43
3.1. Antibacterial Activities of Endolichenic Fungi 43
3.2. Antifungal Activities of Endolichenic Fungi 46
3.3. Chemical Profiles of Chaetomium globosum and Nodulisporium sp. 49
4. Discussions 52
5. Conclusions 55
Chapter Ⅲ. Antimicrobial Properties of Lichen Substances and Identification of the Biosynthetic Gene Cluster for Lobaric Acid Production 56
1. Introduction 57
2. Materials and Methods 60
2.1. Preparation of Purified Lichen Substances 60
2.2. Bacterial and Fungal Plant Pathogens 62
2.3. Paper Disk Diffusion Assay 62
2.4. Determination of Minimal Inhibitory Concentration 63
2.5. Determination of Median Effective Concentration (EC₅₀) 64
2.6. Preparation of Standard Calibration Curve for Lobaric Acid 64
2.7. RNA Extraction and RNA-seq Analysis 65
3. Results 66
3.1. Purification of Lichen Substances 66
3.2. Antibacterial Activities of Lichen Substances 68
3.3. Antifungal Activities of Lichen Substances 70
3.4. Potentials of Lichen Substances as Novel Pesticides 72
3.5. Lobaric Acid Production in Stereocaulon alpinum 75
3.6. Transcriptomics Analysis for Lobaric Acid Production. 77
4. Discussions 80
5. Conclusions 84
Chapter Ⅳ. Identification of the Biosynthetic Gene Cluster for a Red Pigment Cristazarin Produced by a Lichen-Forming Fungus Cladonia metacorallifera 85
1. Introduction 86
2. Materials and Methods 89
2.1. Fungal Isolate and Growth Conditions 89
2.2. Genome Annotation and Biosynthetic Gene Cluster Identification 89
2.3. Identification of Syntenic Gene Clusters 90
2.4. RNA Extraction for RNA-seq 91
2.5. Phylogenetic Analysis 92
3. Results 93
3.1. Gene Expression Profiling of PKS genes in Cladonia metacorallifera 93
3.2. Demarcation of Boundaries for the Cristazarin BGC 96
3.3. Phylogenetic Dereplication of the Biosynthetic Pathway for Cristazarin 100
3.4. A Proposed Biosynthetic Pathway for Cristazarin 104
4. Discussions 106
5. Conclusions 111
Chapter Ⅴ. Functional Characterization of Salazinic Acid BGC in Parmelia sp. KoLRI_050094 112
1. Introduction 113
2. Materials and Methods 116
2.1. Chemical Analysis of Parmelia sp. KoLRI 050094 116
2.2. Biosynthetic Gene Cluster Identification and BGC Network Analysis 117
2.3. Phylogenomic Analysis of 102 Lichen Genomes 117
2.4. Preparation of Protoplasts 118
2.5. Heterologous Expression of Parmelia sp. KoLRI 050094 119
2.6. Validation of the Transformants 123
2.7. Chemical Profiles of Transformants. 125
3. Results 129
3.1. Parmelia sp. KoLRI 050094 Produce Salazinic Acid 129
3.2. PKS1 is Linked with Salazinic Acid Production in Parmelia sp. 132
3.3. Sal3 Converts Depside to a Depsidone 137
3.4. Sal6 Oxidizes an Alcohol to an Aldehyde in the Depsidone Compound 140
4. Discussion 144
5. Conclusions 148
REFERENCES 149
Table 1. Reported biological activities of lichens and their secondary metabolites. 19
Table 2. MIC values of lichen substances for Clavibacter michiganensis subsp. michiganensis. 73
Table 3. Annotation of the lobaric acid biosynthetic gene cluster in Stereocaulon alpinum. 79
Table 4. Annotation of the cristazarin biosynthetic gene cluster in Cladonia metacorallifera. 99
Table 5. List of primers used in the study. 122
Table 6. NMR spectroscopic data (DMSO-d₆) for compound 2. 128
Table 7. Annotation of the PKS1 biosynthetic gene cluster in Parmelia sp. KoLRI_050094. 136
Figure 1. Antibacterial activity of the crude extracts of fourteen ELFs grown in different culture media against Clavibacter michiganensis subsp. michiganensis. 44
Figure 2. Antifungal activity of ELFs cultured in various media. (A) percentage of the antifungal activity exhibited by all cultivated ELF. (B)... 45
Figure 3. Antifungal activity of Chaetomium globosum and Nodulisporium sp. against fourteen plant pathogenic fungi. Each disk contains 40 µl of methanol... 48
Figure 4. LC-MS/MS of Chaetomium globosum. The fifteen compounds were detected in C. globosum extract but those written in bold letters were previously reported to have microbial activity. 50
Figure 5. LC-MS/MS of Nudolisporium sp. The twelve compounds were detected in C. globosum extract but those written in bold letters were previously reported to have microbial activity. 51
Figure 6. HPLC analysis and chemical structures of purified lichen substances. The major peaks in each chromatogram represent the indicated... 67
Figure 7. Antibacterial activity of lichen substances. (A) An example of the culture plate of Clavibacter michiganensis subsp. michiganensis showing... 69
Figure 8. Antifungal activity of lichen substances. Relative antifungal activities of lichen substances against twelve plant pathogenic fungi are... 71
Figure 9. The effective concentration (EC₅₀) of lichen substances against plant pathogenic fungi. Antifungal activities were measured by growth rate... 74
Figure 10. Time course experiment showing the production of lobaric acid (A) Chromatogram of S. alpinum extracts grown in varying culture incubation time. The lichen-forming fungi were cultured in MY media within 3 weeks to 9... 76
Figure 11. Transcriptomic analysis of Stereocaulon alpinum for lobaric acid production. (A). RNA expression of polyketide syntheses in S. alpinum (B) Line graph of the RNA expression of all non-reducing PKS in S. alpinum (C)... 78
Figure 12. Identification of polyketide synthase for the biosynthesis of cristazarin. PKS gene expression profiles of the Cladonia metacorallifera mycobiont... 95
Figure 13. Demarcation of the cristazarin BGC boundaries by mapped reads of RNA-seq. (A) Mapped reads of five RNA-seq samples of the C.... 98
Figure 14. Phylogenetic dereplication of polyketide synthases related to melanin production. A maximum likelihood phylogenetic tree of non-... 102
Figure 15. Divergent biosynthetic routes of cristazarin. A proposed biosynthetic pathway of cristazarin from a polyketide precursor, 2-acetyl-... 105
Figure 16. Biosynthesis of salazinic acid in lichen-forming fungi. HPLC chromatogram of Parmelia sp. KoLRI 21559 lichen thalli showing salazinic... 130
Figure 17. A coalescent-based tree of 102 lichen species. The blue boxes outside the scientific names indicate the presence of PKS1, PKS2, and PKS23... 131
Figure 18. Biosynthetic gene clusters including PKS1 or PKS23 in 102 lichen species. (A) PKS23 and its accessory genes observed in 10 fungal species. The... 135
Figure 19. 4-O-demethylbarbatic acid undergoes oxidation by sal3. (A) Agarose gel electrophoresis after amplification of sal3 gene in gDNA and... 139
Figure 20. Conversion of alcohol to aldehyde in the depsidone molecule. (A) Agarose gel electrophoresis after amplification of sal3 and sal6 genes in the... 142
Figure 21. Chemical profile of atr1-sal3-sal6 strain. (A) Total ion chromatogram of the extract from sal3-sal6 strain, with the three major peaks... 143