Title Page
Abstract
Contents
1. Introduction 15
2. Materials & Methods 20
2.1. Isolation and Whole Genome Sequencing of Streptomyces sp. MMBL 11-1 20
2.2. Strain growth conditions 22
2.3. Antibacterial Activity of Bioassay 22
2.4. Isolation of Biosynthetic Gene Cluster into pESAC-13 23
2.5. RNA sequencing of Streptomyces sp. MMBL 11-1 23
2.6. HPLC analysis to cultivation samples 25
3. Results & Discussion 26
3.1. Optimization of S11-1 plate culture and Bioassay 26
3.2. Whole Genome Sequencing and selection of biosynthetic gene clusters of S11-1 29
3.3. Prediction of Compound Structures Based on Selected BGCs 38
3.4. Isolation of selection three BGC via BAC library construction 41
3.5. RNA sequencing of S11-1 44
3.6. Realization Attempt: Heterologous Expression of BGC 11, BGC 25, and BGC 33 in Streptomyces 46
4. Conclusion 52
5. References 55
Table 1. Genomic features of S11-1 30
Table 2. Analysis of secondary metabolite 37 BGCs by antiSMASH 7.0.1 of S11-1 31
Table 3. Comparing the BGCs of S11-1 and the BGCs of kitacinnamycin compound for similarity 35
Table 4. Comparing the BGCs of S11-1 and the BGCs of massinidine compound for similarity 37
Table 5. Primer list used in this study. 42
Figure 1. Scheme of BGC Exploration and Activation of Streptomyces sp. MMBL 11-1. 19
Figure 2. (A) cultured in various mediums to find the optimal medium for spore formation of S11-1. (B) selected four medium (MS, ISP4, ISP2+... 29
Figure 3. A phylogenetic tree devoid of root was generated to elucidate the evolutionary ties between Streptomyces sp. MMBL 11-1(S11-1) and... 31
Figure 4. Three selected BGCs for the discovery of novelty and antibacterial activity compounds. 35
Figure 5. Prediction of PK-NRP type biosynthesis process to be synthesized by BGC 11 and BGC 25 of S11-1 and structure of... 41
Figure 6. Screening of three BAC colonies. 43
Figure 7. Results of liquid culture analysis in S11-1. 45
Figure 8. Comparing the transcription levels of three BGC genes with the housekeeping gene (hrdA). 46
Figure 9. Each screened BGC was the heterologous expression in a Streptomyces host via conjugation (NC: negative control). 49
Figure 10. Heterologous expression strain's antibacterial activity assay. 50
Figure 11. HPLC results of co-cultivation between S. coelicolor del 4 with heterologous expression of BGC 11 and BGC 25. 51