Genome based analysis for the bioactive potetial of streptomyces gilvosporeus strain f607

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE GENOME-BASED ANALYSIS FOR THE BIOACTIVE POTENTIAL OF STREPTOMYCES GILVOSPOREUS STRAIN F607 HANOI – 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: GENOME-BASED ANALYSIS FOR THE BIOACTIVE POTENTIAL OF STREPTOMYCES GILVOSPOREUS STRAIN F607 Student : VU NGAN HA Department : Biotechnology Supervisor : Dr Dinh Truong Son HANOI – 2021 COMMITMENT I hereby declare that this is my research work The figures and image results, which presented in the thesis, are truthful and have not been published in other scientific studies I hereby certify that all information and references in this thesis have been clearly originated Hanoi, February 24th, 2021 Student Vu Ngan Ha i ACKNOLEDGEMENTS The topic " GENOME-BASED ANALYSIS FOR THE BIOACTIVE POTENTIAL OF Streptomyces gilvosporeus strain F607" is the content that I choose to research and graduate thesis after four years at Faculty of Biotechnology, Vietnam National University of Agriculture During my graduation thesis, I have received a lot of help to complete my thesis First of all, I would like to sincerely thank Dr DINH TRUONG SON, who enthusiastically guided, encouraged and helped me throughout the research process for me to complete this thesis Besides, I would like to sincerely thank teachers in the at the Laboratory of Department of Plant Biotechnology, Faculty of Biotechnology for their valuable comments on the thesis On this occasion, I would also like to thank the Department of Plant Biotechnology, the Faculty of Biotechnology, Vietnam National University of Agriculture, for creating conditions and time for me throughout the research process Finally, I would like to thank my relatives and friends who have always been by my side, encouraging me to complete this course and essay Hanoi, February 24th, 2021 Student Vu Ngan Ha ii CONTENTS COMMITMENT i ACKNOLEDGEMENTS ii LIST OF ABBREVIATIONS v LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT ix PART INTRODUCTION PART LITERATURE REVIEW 2.1 The genus Streptomyces 2.1.1 General characteristics of Streptomyces 2.1.2 Morphological development of Streptomyces 2.2 Secondary Metabolites from Streptomyces 2.3 Introduction to Ficellomycin 2.4 Bioinformatics software 11 2.4.1 Blast2Go 11 2.4.2 antiSMASH 13 PART MATERIALS AND METHODS 14 3.1 Study laboratory and time 14 3.2 Materials 14 3.3 Methods 14 3.3.1 Identification, functional annotation and analysis the S gilvosporeus strain F607 15 3.3.2 Secondary metabolite biosynthesis gene cluster prediction 15 PART RESULT AND DISCUSSION 16 4.1 General genomic features of S gilvosporeus strain F607 16 4.2 Molecular characteristic related to the whole - genome of S gilvosporeus F607 19 4.3 Functional Annotation 22 4.4 Biosynthetic gene clusters for secondary metabolites of strain F607 29 4.5 Proposed biosynthetic pathway of ficellomycin from S gilvosporeus strain F607 33 iii PART V CONCLUSION AND SUGGESTION 39 Conclusion 39 Suggestion 39 REFERENCES 40 APPENDIX 44 iv LIST OF ABBREVIATIONS Abbreviations Definition NCBI National Center for Biotechnology Information COG Clusters of Orthologous Genes rRNA Ribosomal ribonucleic acid tRNA Transfer ribonucleic acid BGCs Biosynthetic gene clusters BLAST Basic local alignment search tool GO Gene ontology CD Median curative dose CDS Coding sequences v LIST OF TABLES Table 4.1 Characteristics of S gilvosporeus strain F607 retrieved from NCBI 16 Table 4.2 The number of gene with functional classification of the whole S gilvosporeus strain F607 genome 18 Table 4.3 List of putative secondary metabolite producing biosynthetic clusters as predicted by antiSMASH 30 Table 4.4 Accession number of ORFs in the ficellomycin biosynthetic gene cluster (Liu et al., 2017) 34 Table Comparison between amino acid sequences with whole genome of S gilvosporeus strain F607 38 vi LIST OF FIGURES Figure 2.1 Three types of living strategies of Streptomyces Figure 2.2 Structures of several industrially exploited secondary metabolites produced by Streptomyces Figure 2.3 Show the life – cycle of Streptomyces (Barka et al., 2016) Figure Various pathways responsible for the assembly of secondary metabolites Figure 2.5 Examples of bacterial secondary metabolites belong to polyketides Figure 2.6 Chemical structures of ficellomycin and aziridine moiety 10 Figure 2.7 Schematic representation of Blast2GO application 12 Figure General contents and pipeline for this study is illustrated in the following schematic diagram 14 Figure 4.1 Circular map of S gilvosporeus strain F607 genome 17 Figure 4.2 Functional classification of the whole S gilvosporeus strain F607 genome 19 Figure 4.3 Overview of data distribution from the BLAST2GO annotation pipeline 20 Figure 4.4 Graphical distribution of the length of the predicted S gilvosporeus F607 20 Figure 4.5 The GO-terms retrieved though the InterProScan results 21 Figure Database Resources of Mapping 22 Figure 4.7 GO level distribution chart for S gilvosporeus strain F607 22 Figure 4.8 Gene ontology (GO) assignment (Level GO terms) for the whole genome of S gilvosporeus strain F607 24 Figure 4.9 Cellular component combined graph of annotation of S gilvosporeus F607 26 Figure 4.10 Molecular function combined graph of annotation of S Gilvosporeus F60727 Figure 4.11 Biological process combined graph of annotation of S gilvosporeus F607 28 Figure 4.12 Distribution of secondary metabolite gene clusters in S gilvosporeus F607 29 Figure 4.13 antiSMASH predicted biosynthetic their predicted core structures 32 Figure 4.14 antiSMASH predicted biosynthetic their predicted biosynthesis gene clusters for three lasso peptide clusters 33 Figure 4.15 Gene cluster was predicted to synthesis ficellomycin of strain F607 33 Figure 4.16 Proposed biosynthetic pathway of ficellomycin (Liu et al., 2017) 35 vii Figure 4.17 Comparison between 15 amino acid sequences with whole genome of S gilvosporeus strain F607 36 Figure 18 Four sequences with same function with fic 36 38 viii Figure 4.16 Proposed biosynthetic pathway of ficellomycin (Liu et al., 2017) 35 Figure 4.17 Comparison between 15 amino acid sequences with whole genome of S gilvosporeus strain F607 N0 ORFs Fic 16 Accession number WP_042180823 Fic 17 Hits * Similarity 50.02% WP_052864002 * 52.87% Fic 18 WP_052863999 * 70.01% Fic 19 BAW27677 * 81.05% Fic 20 BAW27676 * 44.62% Fic 21 WP_061295471 * 53.43% Fic 23 WP_037900779 * 45.42% Fic 24 WP_037900777 * 41.13% Fic 25 WP_043666571 * 54.22% 10 Fic 28 WP_018890853 * 42.26% 11 Fic 29 WP_007383324 * 44.36% 12 Fic 30 WP_018893136 * 42.26% 13 Fic 32 WP_061295458 * 45.46% 14 Fic 36 SBU95134 15 Fic 33 BAW27695 * 40.06% Besides the genes directing AHYC moiety biosynthesis, some unique genes necessary for ficellomycin biosynthesis were also identified from the ficellomycin gene cluster The genes of fic 25 and fic 36 encode proteins, which exhibit high sequence similarity to glutamine – scyllo - inositol transaminase and scyllo-inosamine – phosphate amidinotransferase Given that aminotransferase and an amidinotransferase 36 have been reported to catalyze the transamination and transamidination that yields guanidine in the streptomycin biosynthesis pathway (Ohnuki et al., 1985) Therefore, fic25 and fic 36 were proposed to create ficellomycin’s guanidine group (Fig 4.16) However, fic 36 sequence did not appear in ficellomycin biosynthesis pathway after using local blast in Blast2GO UniProt is well known to be a freely accessible database of protein sequence and functional information It contains a large amount of information about the biological function of proteins derived from the research literature It is developed by the UniProt consortium, which consists of several European bioinformatics organizations and a foundation from Washington, DC, United States We use the Basic Local Alignment Search Tool (BLAST) in UniProt finds similarity sequence with fic 36 sequences, which can be used to infer functional between sequences The results have one amino acid sequences that have a similarity sequence with fic 36 with an identity reached 99.1% The name of protein is Inosamine-phosphate amidinotransferase which belongs to Streptomyces griseus Especially, this protein has an enzyme commission number (EC:2.1.4.2) The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze More clearly, if different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number The search for enzymes with the same catalytic function was performed in the KEGG (Kyoto Encyclopedia of Genes and Genomes) KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies The results have four amino acid sequences which have same function with fic 36 (Figure 4.18) After that, we used NCBI blast+ software to perform Blast searches locally on my PC against a local database The result shows all four amino acid sequences which is appeared to the whole genome of S gilvosporeus strain F607 with sizable similarity (Table 4.5) 37 Figure 18 Four sequences with same function with fic 36 Table Comparison between amino acid sequences with whole genome of S gilvosporeus strain F607 Name sequence Hits Similarity SGR_5933 * 88.55% SBI_00621 * 84.2% SGLAU_01160 * 89.12% EIZ62_06155 * 84.06% From here we can conclude that S gilvosporeus strain F607 can produce the same 15 amino acid sequences above This proves that the pathway can be formed to produce ficellomycin in S gilvosporeus F607 38 PART V CONCLUSION AND SUGGESTION Conclusion This thesis further studied a whole-genome characterization and especially concentrated on the analysis of the bioactive potential of secondary metabolite biosynthetic gene clusters of S gilvosporeus strain F607 by using several bioinformatics tools such as EzBioCloud, AntiSMASH, Blast2GO AntiSMASH analysis reveals 30 putative biosynthetic gene clusters (BGCs) for the whole genome of this strain Therein, some gene cluster is indicated have ability to produce secondary metabolites such as natamycin with high similarity (95%) Annotation of genes was performed against databases such as NCBI nr database, Gene Ontology, KEGG, Uniprot, Interpro A total of 9868 GO terms were annotated including 2025 sequences belong to "Cellular component"; 4747 sequences belong to "Molecular Function", and 3096 sequences belong to "Biological Process" Ficellomycin is chosen to analyze bioactive potential because of its ability to against antibiotic-resistant bacteria Using local blast in Blast2go, there were 14 proposed genes directing AHYC biosynthesis in the ficellomycin pathway were appeared in the genome of S gilvosporeus strain F607 The missing fic 36 was finally found when we used the other four sequences which also annotated the same function with fic 36 for searching against S gilvosporeus strain F607 genome This result allowed us to postulate that S gilvosporeus strain F607 potentially can produce ficellomycin Suggestion The potential of this strain in producing ficellomycin should be further investigated 39 REFERENCES Amin Hasani A.K., Khosrow Issazadeh (2014) Streptomycetes: Characteristics and Their Antimicrobial Activities International journal of Advanced Biological and Biomedical Research Vol pp 12 Anand S., Prasad M.V., Yadav G., Kumar N., Shehara J., Ansari M.Z and Mohanty D (2010) SBSPKS: structure based sequence analysis of polyketide synthases Nucleic Acids Res Vol 38 pp W487-496 Anderson A.S and Wellington E.M (2001) The taxonomy of Streptomyces and related genera Int J Syst Evol Microbiol Vol 51 pp 797-814 Argoudelis 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a review Nat Prod Rep Vol 33 pp 988-1005 43 APPENDIX Additional file 1: Table S1 List of number genes with length of S gilvosporeus strain F607 Lengh (bp) 75 80 90 95 105 110 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 260 No Sequence 3 4 12 12 13 14 15 22 30 13 35 23 14 32 26 12 31 25 17 28 21 11 23 23 Lengh (bp) 270 275 280 285 290 295 300 305 310 315 320 325 330 335 340 345 350 335 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 No Sequence 22 23 12 24 25 16 24 34 15 27 34 17 25 27 12 26 26 20 23 24 17 25 42 12 28 34 42 40 15 38 31 18 38 40 44 Lengh (bp) 450 455 460 465 470 475 480 485 490 495 500 505 510 515 520 525 530 535 540 545 550 555 560 565 570 575 580 585 590 595 600 605 610 615 620 No Sequence 37 35 16 34 39 23 26 32 24 32 21 26 23 25 14 30 35 11 34 27 18 33 23 17 39 38 11 29 39 13 40 30 15 36 38 265 630 635 640 645 650 655 660 665 670 675 680 685 690 695 700 705 710 715 720 725 730 735 740 745 750 755 760 765 770 775 780 785 790 795 800 805 810 815 820 825 830 11 24 38 23 48 43 29 48 33 21 40 38 13 34 23 16 31 25 19 35 24 19 37 34 15 28 39 20 51 37 27 35 35 17 43 31 14 34 41 15 43 30 445 840 845 850 855 860 865 870 875 880 885 890 895 990 995 1000 1005 1010 1015 1020 1025 1030 1035 1040 1045 1050 1055 1060 1065 1070 1075 1080 1085 1090 1095 1100 1105 1110 1115 1120 1125 1130 16 40 49 21 26 32 13 39 35 19 41 31 14 43 24 16 33 17 14 28 35 18 28 24 11 20 27 12 25 23 14 24 26 10 24 17 13 13 26 14 29 25 45 625 1140 1145 1150 1155 1160 1165 1170 1175 1180 1185 1190 1195 1200 1205 1210 1215 1220 1225 1230 1235 1240 1245 1250 1255 1260 1265 1270 1275 1280 1285 1290 1298 1300 1305 1310 1315 1320 1325 1330 1335 1340 19 26 26 17 34 24 19 26 18 18 25 13 15 25 16 22 23 16 26 33 18 22 27 10 23 27 22 22 133 17 18 12 14 17 12 20 26 17 24 835 1350 1355 1360 1365 1370 1375 1380 1385 1390 1395 1400 1405 1410 1415 1420 1425 1430 1435 1440 1445 1450 1455 1460 1465 1470 1475 1480 1485 1490 1495 1500 1505 1510 1515 1520 1525 1530 1535 1540 1545 1550 20 21 14 21 21 17 16 16 12 19 21 20 18 24 17 16 16 16 13 12 11 17 14 15 10 12 12 10 12 11 16 1135 1560 1565 1570 1575 1580 1585 1590 1595 1600 1605 1610 1615 1620 1625 1630 1635 1640 1645 1650 1655 1660 1665 1670 1675 1680 1685 1690 1695 1700 1705 1710 1715 1720 1725 1730 1735 1740 1745 1750 1755 1760 14 11 10 18 12 11 12 12 6 46 1345 1770 1775 1780 1785 1790 1795 1800 1805 1810 1815 1820 1825 1830 1835 1840 1845 1850 1855 1860 1865 1870 1875 1880 1885 1890 1895 1900 1905 1910 1915 1920 1925 1930 1935 1940 1945 1950 1955 1965 1970 1975 12 4 5 6 7 5 5 1555 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030 2035 2040 2045 2050 2055 2060 2065 2070 2075 2085 2090 2095 2100 2105 2110 2115 2120 2125 2130 2135 2145 2150 2155 2160 2165 2170 2180 2185 2190 2195 2200 2 3 1 3 2 1 1 4 10 1 1 1765 2210 2215 2220 2225 2230 2240 2245 2250 2255 2260 2265 2270 2280 2285 2290 2295 2310 2315 2320 2325 2330 2335 2340 2345 2350 2355 2365 2370 2375 2380 2385 2390 2395 2400 2405 2410 2420 2425 2430 2435 2440 2 2 1 2 2 1 2 1 2 1 47 1980 2450 2460 2465 2480 2485 2495 2500 2510 2515 2520 2525 2530 2545 2550 2555 2565 2570 2575 2580 2585 2590 2595 2600 2605 2620 2630 2640 2645 2650 2655 2660 2670 2675 2680 2685 2690 2705 2710 2715 2735 2745 3 2 1 2 1 3 1 1 2205 2755 2760 2765 2775 2780 2790 2795 2805 2820 2825 2835 2845 2850 2855 2865 2870 2875 2885 2890 2895 2910 2915 2920 2925 2930 2940 2945 2955 2965 2970 2975 2980 2990 3000 3020 3025 3050 3060 3080 3090 3110 1 1 2 1 1 1 1 1 1 1 1 1 1 1 2445 3135 3140 3150 3155 3170 3185 3195 3200 3210 3230 3255 3260 3270 3285 3290 3305 3310 3315 3325 3425 3445 3475 3480 3485 3495 3510 3535 3545 3555 3565 3570 3610 3615 3620 3625 3645 3655 3690 3765 3775 3795 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 48 2750 3860 3890 3900 3905 3915 3955 3975 4005 4010 4015 4040 4120 4160 4185 4245 4250 4330 4525 4580 4600 4650 4680 4745 4815 4920 4955 5075 5260 5265 5435 5510 6095 6120 6190 6735 6845 10310 11715 15285 15450 20435 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3125 Additional file 2: Table S2 3845 Process 28455 1 Function Component GO level Annotated gene GO level Annotated gene GO level Annotated gene 0 0 0 1 13 117 269 1435 75 168 640 532 532 1943 1467 508 1514 123 553 792 22 551 383 67 450 729 10 239 10 57 10 11 599 11 15 11 12 195 12 12 13 77 13 13 14 16 14 14 15 15 15 49