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MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ----------------------------- CAO DUC TUAN PROJECT STUDY ON ANTIMICROBIAL SECONDARY METABOLITES ISOLATED FROM SELECTED MARINEDERIVED ACTINOBACTERIA STRAINS BELONG TO STREPTOMYCES GENUS Major: Organic Chemistry Code: 9.44.01.14 SUMMARY OF ORGANIC CHEMISTRY DOCTORAL THESIS HA NOI – 2020 This project was completed at Graduate University Science and Technology – Vietnam Academy of Science and Technology. Scientific Advisor 1: Assoc. Prof. Doan Thi Mai Huong Scientific Advisor 2: Dr. Le Thi Hong Minh Reviewer 1: … Reviewer 2: … Reviewer 3: …. The thesis will be defended at Graduate University of Science and Technology – Vietnam Academy of Science and Technology, at: … The thesis can be found at: - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology. - The National Library of Vietnam. 1 INTRODUCTION 1. Basic of the thesis Natural resources have been used as drugs, additives and poisons for a long time (Marderosian, 1969). To date, natural products, most of them from terrestrial, play a major role in pharmaceutical industries (Harvey, 2000). Although, oceans accounted for about 70% of earth surface, proved to be very high biodiversity, researches on marine natural products only started recently (Fattorusso, 2012). There are over 30,000 marine natural products known to the public with diverse chemical structures and biological activities (Hu, 2011). Some reported marine natural products had structure similar to that of microorganism. It suggested that microorganism may involve in the biosynthesis of these compounds or microorganism might be the production source (Molinski, 2009). Moreover, one of the major advantages of marine microbial research is that it only requires a small amount of natural collected marine samples. Laboratory isolation, fermentation can help to produce enough material for further researches. Vietnam, situated in the tropical Pacific region with high marine biodiversity, has enormous potential in marine resources. Our government also supports the utilization of marine resources for social and economic development (Nguyễn Phú Trọng, 2018). Besides, Vietnam is one of a high antibiotic resistance prevalent country of the word (WHO, 2014). Therefore, development of new antimicrobial reagents domestically is urgently needed. Based on those reasons, project “Study on antimicrobial secondary metabolites isolated from selected marine-derived actinobacteria strains belong to Streptomyces genus” was implemented. 2. Thesis aims Searching for antimicrobial secondary metabolites from actinobacteria isolated from Vietnam marine samples: - Isolation and screening for Vietnam antimicrobial marine-derived actinobacteria strains. - Isolation and structure elucidation of compounds from 2 promising isolated actinobacteria strains. - Determination the antimicrobial activities of isolated compounds. 2 3. Main research activities 1. Isolation and culture actinobacteria strains from collected marine samples; 2. Screening the antimicrobial activities of isolated strains, select two promising strains for identification and fermentation (50L); 3. Isolation of secondary metabolites from 2 fermented strains; 4. Structure elucidation of isolated compounds; 5. Determination of antimicrobial activities of isolated compounds. CHAPTER 1. OVERVIEW 1.1. Marine biological resources 1.2. Actinomycetes 1.2.1. Classification of Actinomycetes 1.2.2. Streptomyces genus 1.2.3. Antibiotics from Streptomyces genus 1.3. Antimicrobial secondary metabolites from Streptomyces genus 1.3.1. World publications 1.3.1.1. Streptomyces sp. isolated from marine sediments 1.3.1.2. Streptomyces isolated from marine invertebrates 1.3.1.3. Streptomyces isolated from other marine sources 1.3.1.4. Antimicrobial secondary metabolites developed by marine Streptomyces sp. genome mining 1.3.2. Vietnam publications CHAPTER 2. MATERIALS AND METHODS 2.1. Materials and equipment 2.2. Research Methods 2.2.1. Sample collection methods 2.2.2. Marine actinobacteria isolation method (Williams, 1965; Williams, 1971; Vũ Thị Minh Đức, 2001) 2.2.3. Marine actinobacteria purification and storage methods (Nguyễn Lân Dũng, 2003) 3 2.2.4. Marine actinobacteria activation and fermentation methods (Vũ Thị Minh Đức, 2001; Nguyễn Lân Dũng, 2003) 2.2.5. Culture broth extraction method (Carlson, 2014; Tanouye, 2015) 2.2.6. Actinobacteria identification methods (Holt, 1989; Sambrook, 1989; Weisburg, 1991; Li, 2007) 2.2.7. Actinobacteria fermentation (50 L) method (Basilio, 2003; Nguyễn Văn Cách, 2004) 2.2.8. Secondary metabolite isolation methods 2.2.9. Structural elucidation methods 2.2.10. Antimicrobial screening method (Hadacek, 2000) CHAPTER 3. EXPERIMENT AND RESULTS 3.1. Sample collection results Using the described equipment and methods, 23 marine samples were collected, including: - 09 samples from the sea of Quang Binh (2 sediments, 3 algae, 3 soft corals and 1 sponge). - 14 samples from Cu Lao Cham, Quang Nam (5 sediments, 2 soft corals, 1 sponge, 2 echinoderms, 2 sea-rabbits, 1 snake tail and 1 invertebrate). 3.2. Actinomycete isolation results Processing 23 marine samples collected at the sea of Quang Binh and Cu Lao Cham, Quang Nam led to the isolation of 35 actinobacteria strains with single colonies of unique morphology and color. 3.3. Antimicrobial activities of isolated actinobacteria strains 35 isolated actinobacteria strains were cultured in small scale (500 mL). Their culture broths were extracted with EtOAc and screened for antimicrobial activities. Obtain results (Table 4.1) showed that 24/35 isolated strain exhibited antimicrobial activities, of which, 4/35 stains were active against at least 4 test microbial pathogens. Two strain (G212 and G278), inhibited 5 tested pathogens, representing two marine areas, were selected for further study. 4 3.4. Identification of strain G212 and G278 3.4.1. Morphological properties of strain G212 and G278 3.4.2. Identification based on 16S rRNA gene sequence 3.4.2.1. Total DNA isolation 3.4.2.2. 16S rRNA gene amplification 3.4.2.3. G212 and G278 16S rRNA gene sequences The 16S rRNA gene sequences of strain G212 and G278, were compared with GeneBank data, in combination with establishing neighbor joining tree. Obtained results indicated that both strains (G212 and G278) had close relationship with members of genus Streptomyces. The 16S rRNA gene sequences of strain G212 and G278 were registered in GeneBank with accession number of MF187963 and MF960781, respectively. 3.5. Fermentation (50 L) of strain G212 and G278 Large scale fermentations were carried to afford fermentation broth (50L) of strain G212 and G278. 3.6. Secondary metabolite isolation from Streptomyces sp. G212 3.6.1. Culture broth extraction The EtOAc (EG212) and n-BuOH (BG212) extracts of 50 L fermentation broth of Streptomyces sp. G212 was achieved with the mass of 2,2 g and 21,4 g, respectively. 3.6.2. Secondary metabolite isolation from extracts of Streptomyces sp. G212 3.6.2.1. Secondary metabolite isolation from EtOAc extract (EG212) From the EtOAc extract (EG212; 2,2 g), 10 compounds were isolated, including G212-1 (5 mg), G212-2 (6 mg), G212-3 (5 mg), G212-4 (7 mg), G212-5 (12 mg), G212-6 (7 mg), G212-7 (6 mg), G212-8 (8 mg), G212-9 (8 mg) and G212-10 (6 mg). 3.6.2.2. Secondary metabolite isolation from n-BuOH extract (BG212) From the n-BuOH extract (BG212; 21,4 g), 6 compounds were isolated, including G212-11 (7 mg), G212-12 (13 mg), G212-13 (5 mg), G212-14 (6 mg), G212-15 (10 mg) and G212-16 (5 mg). 5 3.6.3. Physical parameter and spectral data of Streptomyces sp. G212 isolated compounds 3.6.4. Total synthesis of compound G212-2 and G212-3 To confirm the structure of two new compound 2,4-dichlorophenyl 2,4dichlorobenzoate (G212-2) and 4,5-dihydroxy-7-methyl phthalide (G2123), total synthesis of compound G212-2 and G212-3 was achieved from the commercially available starting material as 2,4-dichlorobenzoyl chloride and 2,3-dimethoxybenzoic acid, respectively. 3.6.4.1. Synthesis of 2,4-dichlorophenyl 2,4-dichlorobenzoate (G212-2 TH) 3.6.4.2. Synthesis of 4,5-dihydroxy-7-methyl phthalide (G212-3) and isomer G212-6’ 3.7. Secondary metabolite isolation from Streptomyces sp. G278 3.7.1. Culture broth extraction The EtOAc (EG278) and n-BuOH (BG278) extracts of 50 L fermentation broth of Streptomyces sp. G278 was achieved with the mass of 3,26 g and 22,4 g, respectively. 3.7.2. Secondary metabolite isolation from extracts of Streptomyces sp. G278 3.7.2.1. Secondary metabolite isolation from EtOAc extract (EG278) From the EtOAc extract (EG278; 3,26 g), 6 compounds were isolated, including G278-1 (6 mg), G278-2 (7 mg), G278-3 (5 mg), G278-4 (10 mg), G278-5 (6 mg), G278-6 (6 mg). 3.7.2.2. Secondary metabolite isolation from n-BuOH extract (BG278) From the n-BuOH extract (BG278; 22,4 g), 10 compounds were isolated, including G278-7 (7 mg), G278-8 (6 mg), G278-9 (5 mg), G27810 (8 mg), G278-11 (6 mg), G278-12 (20 mg), G278-13 (6 mg), G278-14 (10 mg), G278-15 (8,5 mg), G278-16 (5 mg). 3.7.3. Physical parameter and spectral data of Streptomyces sp. G278 isolated compounds 3.8. Antimicrobial activities of isolated compounds (Table 4.33 and Table 4.34) 6 CHAPTER 4. DISCUSSION OF RESULTS 4.1. Sample collection There were 23 collected marine sample, including 9 samples from Quang Binh and 14 samples from Cu Lao Cham, Quang Nam. The number and type of marine samples collected in each area are different, might be reflecting the differences in local biodiversity at the time of sample collection. 4.2. Actinomycete isolation From 9 marine samples collected at the sea of Quang Binh and 14 marine samples collected at the sea of Cu Lao Cham, Quang Nam, 15 and 20 actinobacteria strains were isolated, respectively. 4.3. Antimicrobial activities of isolated actinobacteria strains Antimicrobial screening results of EtOAc extract of 35 isolated actinobacteria strains (Table 4.1) revealed that 24/35 (68,6 %) strains exhibited antimicrobial activities. Of which, 4/35 (11,4 %) strains active against at least 4 tested microorganisms; 11/35 (31,4 %) strongly active against Candida albicans (MIC 2 – 64 µg/mL). Besides, there were 15/35 (42,8%) strain inhibited the growth of Gram positive bacteria while only 6/35 (17,1%) strains inhibited the growth of Gram negative bacteria. Table 4.1. The MIC (µg/mL) of isolated actinobacteria strains. (* Only active strains were presented) Minimum Inhibitory Concentration MIC (µg/mL) No Strain (*) Gram positive E. faecalis S. aureus B. cereus Gram negative E. coli P. aeruginosa S. enterica fungus C.albicans ATCC29212 ATCC25923 ATCC14579 ATCC25922 ATCC27853 ATCC13076 ATCC10231 1 2 3 4 5 6 7 15 actinobacteria strains isolated from Quang Binh sea G183 128 G193 G196 64 G197 128 G202 128 G207 128 G212 128 128 64 256 - 64 - 256 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 G214 128 G216 256 20 actinobacteria strains isolated from Cu Lao Cham, Quang Nam sea G274 256 G275 G276 256 8 G277 256 G278 256 32 16 16 2 G280 256 256 16 32 32 G283 128 G284 256 256 G285 32 G288 32 G289 32 G290 32 16 8 16 G291 128 G292 32 G293 256 S 256 256 128 32 256 128 C 32 (S: Streptomycine; C: Cyclohexamide; (-): MIC > 256 µg/mL) 4.4. Identification of strain G212 and G278 The 16S rRNA gene sequence of strain G212 were 99,57% similar to that of Streptomyces caelestis JS-5 (GeneBank accession no. EU124773) and 99,64% similar to Streptomyces caelestis JS-4 (GeneBank accession no. EU124772). The 16S rRNA gene sequence of strain G278 were 99,86% similar to that of Streptomyces sp. R1 (GeneBank accession no. MK757961) 4.5. Fermentation (50 L) of strain G212 and G278 The large-scale fermentations (50 L) of strain G212 and G278 were successfully performed to afford required materials for next research step. 4.6. Structure elucidation of secondary metabolites isolated from Streptomyces sp. G212 From the culture broth of Streptomyces sp. G212, the isolation and structure elucidation of 16 compounds was achieved (Figure 4.31), including 8 two new compounds (G212-2 and G212-3) and one compound isolated first time from nature (G212-1). These isolated compounds were identified as 1 polyethylene terephthalate: ethylene terephthalate cyclic trimer (G212-1), 1 dichlorophenyl derivative: 2,4-dichlorophenyl 2,4-dichlorobenzoate (G2122), 1 phthalite derivative: 4,5-dihydroxy-7-methyl phthalide (G212-3), 2 antibiotics: germicidine A (G212-4), germicidine B (G212-5), 1 benzopyridine derivative: 3,4-dihydroxy-6,7-dimethyl-quinolin-2carboxylic (G212-9), 3 cyclopeptide: cyclo-(Pro-Val) (G212-10), cyclo(Pro-Tyr) (G212-11), cyclo-(Leu-trans-4-hydroxy-Pro) (G212-12), 2 nucleic acid derivative: 2’-deoxythymidine (G212-7), 2’-deoxyuridine (G212-8), 2 indole: N-[2-(1H-indol-3-yl)-2-oxo-ethyl] acetamide (G21213), indole-3-carboxylic acid (G212-14) and 5-hydroxymethyl-4-hydroxy2,4-dimethyl-2-cyclopentenone (G212-6), 1H-pyrrole-2-carboxylic acid (G212-15), 2-phenylacetic acid (G212-16). Structure of two new compounds, G212-2 and G212-3, were confirmed by total synthesis. Figure 4.31. Compounds isolated from G212