Progress in Microbes and Molecular Biology Genome Report 1 Whole genome sequence of Streptomyces colonosanans strain MUSC 93JT isolated from mangrove forest in Malaysia Hooi-Leng Ser1†, Jodi Woan-Fei Law1†, Wen-Si Tan2, Wai-Fong Yin3, Kok-Gan Chan3,4* 1Novel Bacteria and Drug Discovery (NBDD) Research Group, Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia. 2Illumina Singapore Pte Ltd, Woodlands Industrial Park E1, Singapore. 3Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 4Vice Chancellor Office, Jiangsu University, Zhenjiang 212013, PR China. †These authors contributed equally to the work. Abstract: Under the family Actinobacteria, streptomycetes are ubiquitous in nature, producing a wide spectrum of bioactive compounds including antibacterial, antioxidant, anticancer and immunomodulatory properties. During a screening programme in Malaysia, Streptomyces colonosanans MUSC 93JT was isolated as a novel Streptomyces sp. from the mangrove soil in Sarawak. The strain exhibited potent antioxidant activities and cytotoxic activity against several human cancer cell lines. Due to these data, the strain was subjected to whole genome sequencing to uncover its genomic potential and further improve the understanding of the strain. The genome of MUSC 93JT consists of 7,015,076 bp (G + C content of 69.90%), carrying a total of 5,859 protein coding genes. Analysis using a bioinformatics tool, antiSMASH predicted a total of four biosynthetic gene clusters which displayed similarity of more than 70% to known gene clusters and one of which was associated with the production of a natural protectant, ectoine. Displaying selective toxicity that kills only cancer cells, ectoine has showed its potential to be developed as therapeutic agents for humans. Altogether, the current project clearly highlights the importance of under-explored environment like mangrove in natural product discovery. The availability of whole genome sequence MUSC 93JT warrants subsequent in-depth investigation and optimization for the production of bioactive compounds which can be exploited for the health and wellbeing of mankind. Keywords: Streptomyces; anti-cancer; mangrove; genome; MUSC 93JT; actinobacteria Received: 18th February 2020 Accepted: 20th March 2020 Published Online: 25th March 2020 Citation: Ser H-L, Law JW-F, Tan W-S, et al. Whole genome sequence of Streptomyces colonosanans strain MUSC 93JT isolated from mangrove forest in Malaysia. Prog Microbes Mol Biol 2020; 3(1): a0000061. https://doi.org/10.3687/pmmb/ a0000061 Short Introduction Streptomycetes are filamentous bacteria that can be found in various ecosystems and most well-known for their abil- ity to produce secondary metabolites which can be ex- ploited for the benefits of mankind[1–7]. For instance, the isolation of streptomycin from Streptomyces griseus de- scribed by Professor Waksman and his team was a major breakthrough back in the 1950s, being the first effective treatment against the causative agent of the great white plague, Mycobacterium tuberculosis[8,9]. Even though more than 60 years have passed, drug discovery studies investigating bioactive potential of Streptomyces sp. from various habitat did not regress, but more efforts are now being poured into the investigation of their genom- ic potential[10–19]. Streptomyces colonosanans MUSC 93JT was recovered from mangrove forest soil located at Sarawak, Malaysia during a screening programme for bioactive streptomycetes[10,20]. Forming light yellow aer- ial and vivid yellow substrate mycelium on ISP 2 agar which is a typical trait of streptomycetes, MUSC 93JT was designated as novel species of genus Streptomyces which is closely related to Streptomyces malachitofus- cus NBRC 13059T (99.2% sequence similarity), Strep- Copyright © 2020 by Ser H-L and HH Publisher. This work under licensed under the Creative Commons Attribution-NonCommercial 4.0 International Lisence (CC-BY-NC4.0) *Correspondence: Kok-Gan Chan, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; kokgan@um.edu.my 2 tomyces misionensis NBRC 13063T (99.1%), and Strep- tomyces phaeoluteichromatogenes NRRL 5799T (99.1%) based on phylogenetic analysis using their 16S rRNA genes. Nonetheless, fermentative extracts of MUSC 93JT displayed potent antioxidant activity and anticancer ac- tivity against several human colon cancer cell lines with- out significant cytotoxic effect against human normal co- lon cells. The type strain for MUSC 93JT is available at two culture collection centres with accession of (= DSM 102042T = MCCC 1K02298T). Based on the biosyste- matics study using a polyphasic approach, the strain was selected for whole genome sequencing to explore its ge- nomic potential, particularly the production of bioactive compounds that are responsible for its anticancer and an- tioxidant activities[10,21,22]. Data description Genomic DNA of MUSC 93JT was obtained using Mas- terpure™ DNA purification kit (Epicentre, Illumina Inc., Madison, WI, USA) and subjected to RNase (Qia- gen, USA) treatment[23–25]. Following that, DNA quality check was conducted with NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a Qubit version 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Construction of DNA library was done using Nextera™ DNA Sample Preparation kit (Nextera, USA) and the library quality was checked by Bioanalyzer 2100 high sensitivity DNA kit (Agilent Technologies, Palo Alto, CA). Paired-end sequencing was performed on MiSeq platform with MiSeq Reagent Kit 2 (2 × 250 bp; Illumina Inc., Madison, WI, USA)[26,27]. After trimming, the paired-end reads were de novo assembled on CLC Genomics Workbench version 7 (CLC bio, Denmark), which resulted in 166 contigs and an N50 contig size of approximately 99,963 bp. The genome size of MUSC 93JT comprised 7,015,076 bp, with an average coverage of 53.0-fold and G + C content of 69.90 %. The genome sequence of MUSC 93JT has been deposited at DDBJ/ EMBL/GenBank under accession of MLYP00000000. Table 1. General genomic features of Streptomyces colonasanans MUSC 93JT. Streptomyces colonasanans MUSC 93JT Genome size (bp) 7,015,076 Contigs 166 Contigs N50 (bp) 99,963 G + C content % 69.90 Genome coverage 53.0x Protein coding genes 5,859 tRNA 66 rRNA (5S, 16S, 23S) 3, 1, 1 The assembled genome was annotated using Rapid An- notation using Subsystem Technology (RAST)[28]. Gene prediction was performed using Prodigal version 2.6, while ribosomal RNA (rRNA) and transfer RNA (tRNA) were predicted using RNAmmer and tRNAscan SE version 1.21, respectively[29–31]. The analysis from RAST revealed 5,859 protein-coding genes, along with a total of 71 RNA genes (Figure 1). Based on RAST system, most of the protein-coding genes were shown to be involved in amino acids and derivatives metabolism (9.18%), followed by carbohydrates metabolism (6.21%) and protein metabolism subsystems (4.91%). Further analysis on antibiotics & Secondary Metabolite Analysis SHell (antiSMASH) detected presence of 23 biosynthetic gene clusters in MUSC 93JT genome using “strict” detection settings (version 5.1.1)[32,33]. Among the four biosynthetic gene clusters which displayed similarity of more than 70% to known gene clusters, one cluster was associated with the production of ectoine (75 % gene similarities). Ectoine is commonly expressed by bacteria to survive in harsh environments, protecting these microorganisms against extreme osmotic stress[34–38]. As a compatible solute, ectoine has been shown to be safe as it does not interfere with the host’s metabolism while offering some beneficial effects including antioxidant and protection against ionizing radiation[39–42]. Apart from that, a recent study by Sheikhpour et al. (2019) showed that ectoine induced apoptosis in lung cancer cells without affecting normal cells. As a natural protectant, ectoine seems to be a promising protective agent to be developed for human use, particularly against chronic inflammatory diseases and cancer[43,44]. On top of that, there has been many stud- ies reported ectoine-based spray or lozenges showed su- perior efficacy in treating acute pharyngitis and/or laryngitis, proposing its potential use as adjuvant treatment for an- ti-inflammatory or anti-infective drugs[45,46]. The detec- tion of this biosynthesis gene cluster within the genome of MUSC 93JT reflects the bioactive potential of mangrove-derived actinobacteria (including rare actino- mycetes and streptomycetes and further highlighting the possible development of this strain as “mini-factories” for the production of protective molecule like ectoine[47–49]. With the emerging role of probiotics in regulating human diseases caused by gut dysbiosis (i.e. imbalance in gut microbial population), ectoine as a osmoprotectant could potentially increase the viability of probiotics in food and prolong its shelf life[50–60]. With the availability of the whole genome sequence of MUSC 93JT, these data would greatly accelerate the medium optimization process and allow genomic manipulations to maximize the production of bioactive compounds including ectoine. Conflict of interest The authors declare that there is no conflict of interest in this work. Acknowledgement This work was supported by the University of Malaya for High Impact Research Grant (UM-MOHE HIR Na- ture Microbiome Grant No. H-50001-A000027 and No. A000001-5001) and PPP Grant (PG090-2015B) awarded to K-GC. MUSC 93T genome... 3 Reference 1. Bérdy J. Thoughts and facts about antibiotics: Where we are now and where we are heading. J Antibiotics 2012; 65(8): 385. 2. Subramani R and Aalbersberg W. Marine actinomycetes: an ongoing source of novel bioactive metabolites. Microbiol Res 2012; 167(10): 571–580. 3. Ser HL, Tan WS, Ab Mutalib NS, et al. Genome sequence of Strepto- myces pluripotens MUSC 135T exhibiting antibacterial and antioxidant activity. Mar Gen 2015; 24:281–283. 4. Sangkanu S, Rukachaisirikul V, Suriyachadkun C, et al. Evaluation of antibacterial potential of mangrove sediment-derived actinomycetes. Microbial Pathogen 2017; 112: 303–312. 5. Law JW, Ser HL, Khan TM, et al. The potential of Streptomyces as biocontrol agents against the rice blast fungus, Magnaporthe oryzae (Pyricularia oryzae). Front Microbiol 2017; 8:3. 6. Dhakal D, Pokhrel AR, Shrestha B, et al. Marine rare actinobacteria: Isolation, characterization, and strategies for harnessing bioactive com- pounds. Front Microbiol 2017; 8: 1106. 7. Sakula A. Selman Waksman (1888–1973), discoverer of streptomycin: A centenary review. Brit J Dis Chest 1988; 82: 23–31. 8. Tan LT, Lee LH, and Goh BH. The bioprospecting of anti-Vibrio Strep- tomyces species: Prevalence and applications. Prog Microbes Mol Biol 2019; 2(1). 9. Waksman SA, Reilly HC, and Johnstone DB. Isolation of streptomycin- producing strains of Streptomyces griseus. J Bacteriol 1946; 52(3): 393. 10. Law JW, Ser HL, Duangjai A, et al. Streptomyces colonosanans sp. nov., a novel actinobacterium isolated from Malaysia mangrove soil exhibiting antioxidative activity and cytotoxic potential against human colon cancer cell lines. Front Microbiol 2017; 8: 877. 11. Lee LH, Zainal N, Azman AS, et al. Diversity and antimicrobial ac- tivities of actinobacteria isolated from tropical mangrove sediments in Malaysia. Sci World J 2014; 2014. 12. Jose PA, and Jha B. Intertidal marine sediment harbours Actinobacteria with promising bioactive and biosynthetic potential. Sci Rep 2017; 7(1): 10041. 13. Ser HL, Tan LT, Law JW, et al. Focused review: Cytotoxic and anti- oxidant potentials of mangrove-derived Streptomyces. Front Microbiol 2017; 8: 2065. 14. Qin S, Li WJ, Dastager SG, et al. Actinobacteria in special and extreme habitats: Diversity, function roles, and environmental adaptations. Front Microbiol 2016; 7: 1415. 15. Ser HL, Law JW, Chaiyakunapruk N, et al. Fermentation conditions that affect clavulanic acid production in Streptomyces clavuligerus: A systematic review. Front Microbiol 2016; 7: 522. 16. Tan LT, Chan KG, Chan CK, et al. Antioxidative potential of a Strep- tomyces sp. MUM292 isolated from mangrove soil. BioMed Res Int 2018; 2018. 17. Tan LT, Chan KG, Pusparajah P, et al. Mangrove derived Streptomyces sp. MUM265 as a potential source of antioxidant and anticolon-cancer agents. BMC Microbiol 2019; 19(1): 38. 18. Ser HL, Yin WF, Chan KG, et al. Antioxidant and cytotoxic potentials of Streptomyces gilvigriseus MUSC 26T isolated from mangrove soil in Malaysia. Prog Microbes Mol Biol 2018; 1(1). 19. Ghosh S, Kuisiene N, and Cheeptham N. The cave microbiome as a source for drug discovery: Reality or pipe dream?. Biochem Pharm 2017; 134:18–34. 20. Law JW, Ser HL, Ab Mutalib NS, et al. Streptomyces monashensis sp.nov., a novel mangrove soil actinobacterium from East Malaysia Ser H-L et al. with anti-oxidative potential. Sci Rep 2019; 9(1): 3056. 21. Law JW, Tan KX, Wong SH, et al. Taxonomic and characterization methods of Streptomyces: A review. Prog Microbes Mol Biol 2018; 1(1). 22. Kämpfer P and Glaeser SP. Prokaryotic taxonomy in the sequencing era–the polyphasic approach revisited. Environ Microbiol 2012; 14(2): 291–317. 23. Ser HL, Tan WS, Cheng HJ, et al. Draft genome of amylolytic actinobac- terium, Sinomonas humi MUSC 117T isolated from intertidal soil. Mar Gen 2015; 24: 209–210. 24. Ser HL, Ab Mutalib NS, Yin WF, et al. Genome sequence of Streptomyces antioxidans MUSC 164T isolated from mangrove forest. Prog Microbes Mol Biol 2018. 25. Ser HL, Tan WS, Cheng HJ, et al. Draft genome of starch-degrading actino- bacterium, Microbacterium mangrovi MUSC 115T isolated from intertidal sediments. Prog Drug Dis Biomed Sci 2018. 26. Ser HL, Tan WS, Ab Mutalib NS, et al. Genome sequence of Streptomyces mangrovisoli MUSC 149T isolated from intertidal sediments. Braz J Micro- biol 2018; 49(1): 13–15. 27. Letchumanan V, Ser HL, Tan WS, et al. Genome sequence of Vibrio para- haemolyticus VP152 strain isolated from Penaeus indicus in Malaysia. Front Microbiol 2016; 7: 1410. 28. Aziz RK, Bartels D, Best AA, et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genomics 2008; 9: 75. 29. Lowe TM, Eddy SR. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nuc Acids Res 1997; 25: 955–964. 30. Lagesen K, Hallin P, Rodland EA, et al. RNAmmer: Consistent and rapid annotation of ribosomal RNA genes. Nuc Acids Res 2007; 35: 3100–3108. 31. Hyatt D, Chen GL, Locascio PF, et al. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinform 2010; 11: 119. 32. Blin K, Wolf T, Chevrette MG, et al. antiSMASH 4.0—improvements in chemistry prediction and gene cluster boundary identification. Nuc Acids Res 2017; 45(W1): W36–41. 33. Blin K, Shaw S, Steinke K, et al. antiSMASH 5.0: Updates to the secondary metabolite genome mining pipeline. Nuc Acids Res 2019; 47(W1): W81–87. 34. Czech L, Hermann L, Stöveken N, et al. Role of the extremolytes ectoine and hydroxyectoine as stress protectants and nutrients: Genetics, phyloge- nomics, biochemistry, and structural analysis. Genes 2018; 9(4): 177. 35. He YZ, Gong J, Yu HY, et al. High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli. Microbial Cell Factories 2015; 14(1): 55. 36. Bremer E, Richter AA, Mais CN, et al. Biosynthesis of the stress-protectant and chemical chaperon ectoine: biochemistry of the transaminase EctB. Front Microbiol 2019; 10: 2811. 37. Jebbar M, von Blohn C, and Bremer E. Ectoine functions as an osmoprotec- tant in Bacillus subtilis and is accumulated via the ABC-transport system OpuC. FEMS Microbiol Lett 1997;154(2): 325–330. 38. Schwibbert K, Marin-Sanguino A, Bagyan I, et al. A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elon- gata DSM 2581T. Environ Microbiol 2011; 13(8): 1973–1994. 39. Hahn MB, Meyer S, Schröter MA, et al. DNA protection by ectoine from ionizing radiation: molecular mechanisms. Physical Chem Chem Physics 2017; 19(37): 25717–25722. 40. Rieckmann T, Gatzemeier F, Christiansen S, et al. The inflammation- reducing compatible solute ectoine does not impair the cytotoxic effect of ionizing radiation on head and neck cancer cells. Sci Rep 2019; 9(1): 1–8. 41. Brands S, Schein P, Castro-Ochoa KF, et al. Hydroxyl radical scavenging of the compatible solute ectoine generates two N-acetimides. Arch Bio- Figure 1. Subsystem category distribution of Streptomyces colonosanans MUSC 93JT (based on RAST annotation server). 4 chem Biophysics 2019; 674: 108097. 42. Shaikhpour M, Sadeghi A, Yazdian F, et al. Anticancer and apoptotic ef- fects of ectoine and hydroxyectoine on non-small cell lung cancer cells: An in-vitro investigation. Multidis Cancer Invest 2019; 3(2): 14–19. 43. Bownik A and Stępniewska Z. Ectoine as a promising protective agent in humans and animals. Arch Indust Hygiene Toxicol 2016; 67(4): 260–265. 44. Graf R, Anzali S, Buenger J, et al. The multifunctional role of ectoine as a natural cell protectant. Clinics Dermatol 2008; 26(4): 326–333. 45. Müller D, Lindemann T, Shah-Hosseini K, et al. Efficacy and tolera- bility of an ectoine mouth and throat spray compared with those of saline lozenges in the treatment of acute pharyngitis and/or laryngitis: a prospective, controlled, observational clinical trial. Euro Arch Oto- Rhino-Laryngology 2016; 273(9): 2591–2597. 46. Dao VA, Overhagen S, Bilstein A, et al. Ectoine lozenges in the treatment of acute viral pharyngitis: a prospective, active-controlled clinical study. Euro Arch Oto-Rhino-Laryngology 2019; 276(3): 775–783. 47. Ser HL, Yin WF, Chan KG, et al. Genome sequence of Novosphingobium malaysiense strain MUSC 273T, novel alpha-proteobacterium isolated from intertidal soil. Prog Microbes Mol Biol 2018; 1(1). 48. Tan LT, Chan KG, Lee LH, et al. Streptomyces bacteria as potential pro- biotics in aquaculture. Front Microbiol 2016; 7:79. 49. Azman AS, Othman I, S Velu S, et al. Mangrove rare actinobacteria: tax- onomy, natural compound, and discovery of bioactivity. Front Microbiol 2015; 6: 856. 50. Kumar R, Sood U, Gupta V, et al. Recent advancements in the devel- opment of modern probiotics for restoring human gut microbiome dys- bio-sis. Ind J Microbiol 2019: 1–4. 51. Yang D, Zhao D, Shah SZ, et al. Implications of gut microbiota dys- biosis and metabolic changes in prion disease. Neurobiol Dis 2020; 135: 104704. 52. Lee LH, Ser HL, Khan TM, et al. IDDF2018-ABS-0239 Dissecting the gut and brain: Potential links between gut microbiota in develop- ment of alzheimer’s disease? Gut 2018; 67(2). 53. Kappel BA and Lehrke M. Microbiome, diabetes and heart: A novel link?. Herz 2019; 44(3): 223–230. 54. Li K, Hu Z, Ou J, and Xia K. Altered gut microbiome in autism spectrum disorder: Potential mechanism and implications for clinical intervention. Glob Clin Transl Res 2019; 1: 45–52. 55. Lee LH, Ser HL, Khan T, et al. Relationship between autism and gut microbiome: Current status and update. Gut 2019; 68. 56. Viljanen M, Savilahti E, Haahtela T, et al. Probiotics in the treatment of atopic eczema/dermatitis syndrome in infants: A double‐blind placebo‐controlled trial. Allergy 2005; 60(4): 494–500. 57. Dharmaraj S, Dhevendaran K. Evaluation of Streptomyces as a probi- otic feed for the growth of ornamental fish Xiphophorus helleri. Food Tech Biotechnol 2010; 48(4): 497–504. 58. Norouzi H, Danesh A, Mohseni M, et al. Marine actinomycetes with probiotic potential and bioactivity against multidrug-resistant bacte- ria. Int J Mol Cell Med 2018; 7(1): 44. 59. Omara AM, Sharaf AE, Atef A, et al. Optimizing ectoine biosynthesis using response surface methodology and osmoprotectant applica- tions. Biotechnol Lett 2020: 1–5. 60. Lee LH, Chan KG, Stach J, et al. The search for biological active agent(s) from actinobacteria. Front Microbiol 2018; 9: 824. MUSC 93T genome...