Outlook of fosmid library from metagenomic of the symbionts associated with coral Siderastrea stellata: structural and functional screening for metabolic and antimicrobial activity / Perspectiva da biblioteca fosmidial a partir da metagenômica de simbiontes associados ao coral Siderastrea stellata: triagem estrutural e funcional da atividade metabólica e antimicrobiana


  • Moara Silva Costa Brazilian Journals Publicações de Periódicos, São José dos Pinhais, Paraná
  • Rachel Passos Rezende
  • Cristiane de Araújo Quinto
  • Eric de Lima Silva Marques
  • Carlos Priminho Pirovani
  • Bianca Mendes Maciel
  • Maria Clara Bessa Souza
  • João Carlos Teixeira Dias




Biocompound, Cnidaria, Heterologous expression, Proteomics.


Microorganisms colonize the corals and produce compounds with interesting biological properties for biotechnology. The aim of the present study was to search for biocompounds with industrial potential through the cloning metagenomic DNA of symbionts the coral S. stellata in fosmid vector. The metagenomic library was analyzed using functional screening and sequencing via the Illumina MiSeq. Of the 3648 clones, eight were identified as proteolytic and six as amylolytic. The proteolytic clone, P07H3, also exhibited antimicrobial activity against S. aureus, S Enterica PT11, S Enterica PT4 and S Typhi. No clones were positive for lipase/esterase. Beta-lactamase was a single subsystem identified by Rapid Annotation using Subsystem Technology (RAST) through fosmid sequencing of clone P07H3. However, there were identifications of enzymes that participate in metabolic processes such as polysaccharide catabolism, oxidation-reduction, signal transduction and phosphorylation. When the hypothetical proteins were re-analyzed they exhibited a defined functional domain. All the identified open reading frames (ORFs) had low identity with proteins deposited in BLASTp. The sequencing shows that most of the genomic fragments of clone P07H3 possibly consist of new proteins. The Proteobacteria phylum had the greatest predominance among the analyzed fragments. This is the first report on the prospection of amylases, proteases and antimicrobials of coral S. stellata samples. The analyzes of this study help to improve the knowledge about the metabolic diversity of S. stellata still little explored and viable in the future study of the identified compounds.


Agnew, C.; Borodina, E.; Zaccai, N.R.; Conners, R.; Burton, N.M.; Vicary, J.A.; Cole, D.K.; Antognozzi, M.; Virji, M.; Brady, R.L. Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1 binding. PNAS. 2011, 108: 15174–15178.

Amann, R.I,; Luswig, W.; Schleifer, K.H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 1995, 59: 143–169.

Assis, D.A.M.; Rezende, R.P.; Dias, J.C.T. Use of Metagenomics and Isolation of Actinobacteria in Brazil’s Atlantic Rainforest Soil for Antimicrobial Prospecting. ISRN Biotechnol. 2014, 1–7.

Aziz, R.K.; Bartels, D.; Best, A.A.; Dejongh, M.; Disz, T.; Edwards, R.A.; Formsma, K.; Gerdes, S.; Glass, E.M.; Kubal, M.; et al. The RAST Server?: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008, 15: 1–15.

Cheng, S.; Wen, Z.; Chiou, S.; Tsai, C.; Wang, S.; Hsu, C.; Dai, C. Ceramide and Cerebrosides from the Octocoral Sarcophyton ehrenbergi. J. Nat. Prod. 2009, 72: 465–468.

Correa, H.; Aristizabal, F.; Duque, C.; Kerr R. Cytotoxic and antimicrobial activity of pseudopterosins and secopseudopterosins isolated from the octocoral Pseudopterogorgia elisabethae of San Andrés and Providencia Islands (Southwest Caribbean Sea). Mar. Drugs. 2011, 9: 334–344.

Costa, C.F.; Amaral F.D.; Sassi R. Branqueamento em Siderastrea Stellata (Cnidaria, Scleractinia) Da Praia De Gaibu, Pernambuco, Brasil. Rev. Nord. Biol. 2001, 15: 15–22.

Craik, C.S.; Page, M.J.; Madison, E.L. Proteases as therapeutics. Biochem. J. 2011, 435: 1–16.

Daniel, R. The metagenomics of soil. Nat. Rev. Microbiol. 2005, 3: 470–478.

Dhanjal, S.D.; Sharma, D. Microbial Metagenomics for Industrial and Environmental Bioprospecting: The Unknown Envoy. Microbial Bioprospecting for Sustainable Development. 2018, 327–352.

Doma?-Pytka, M.; Bardowski, J. Pullulan Degrading Enzymes of Bacterial Origin. Crit. Ver. Microbiol. 2004, 30: 107–121.

ElAhwany, A.M.D.; Ghozlan, H.A., ElSharif, H.A.; Sabry, S.A. Phylogenetic diversity and antimicrobial activity of marine bacteria associated with the soft coral Sarcophyton glaucum. J. Basic. Microbiol. 2015, 55: 2–10.

Gabor, E.M.; Alkema, W.B.L.; Janssen, D.B. Quantifying the accessibility of the metagenome by random expression cloning techniques. Environ. Microbiol. 2004, 6: 879–886.

González, J.M.; Moran, M.A. Numerical dominance of a group of marine bacteria in the ?-subclass of the class Proteobacteria in coastal seawater. Appl. Environ. Microbiol. 1997, 63:4237–4242.

Guillén, D.; Sánchez, S.; Rodríguez-Sanoja, R. Carbohydrate-binding domains: multiplicity of biological roles. Appl. Microbiol. Biotech. 2009, 85:1241–1249.

Gupta, R.; Beg, Q.; Lorenz, P. Bacterial alkaline proteases: Molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 2002, 59:15–32.

Handelsman, J.; Rondon, M.R.; Brady, S.F.; Clardy, J.; Goodman, R.M. Molecular biological access to the chemistry of unknown soil microbes: A new frontier for natural products. Chem. Biol. 1998, 5.

Kanehisa, M. The KEGG databases at GenomeNet. Nucleic Acids Res. 2002, 30: 42–46.

Koh, E.G.L. Do scleractinian corals engage in chemical warfare against microbes? J. Chem. Ecol. 1997, 23: 379–398.

Lämmle, K.; Zipper, H.; Breuer, M.; Hauer, B.; Buta, C.; Brunner, H.; Rupp, S. Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning. J. Biotechnol. 2007, 127: 575–592.

Lee, D.G.; Jeon, J.H.; Jang, M.K.; Kim, N.Y.; Lee, J.H.; Lee, J.H.; Kim, S.J.; Kim, G. Do; Lee, S.H. Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library. Biotechnol. Lett. 2007, 29: 465–472.

Leão, Z.M.A.N.; Kikuchi, R.K.P.; Testa, V. Corals and coral reefs of Brazil. Lat. Am. Coral Reefs 2003, 9–52.

Liu, Y.; Lei, Y.; Zhang, X.; Gao, Y.; Xiao, Y.; Peng, H. Identification and Phylogenetic Characterization of a New Subfamily of ?-Amylase Enzymes from Marine Microorganisms. Mar. Biotechnol. 2012, 14: 253–260.

Mariottini, G.L.; Grice, I.D. Antimicrobials from cnidarians. A new perspective for anti-infective therapy? Mar. Drugs 2016, 14: 1–19.

Naumoff, D.G. GH97 is a new family of glycoside hydrolases, which is related to the ?-galactosidase superfamily. BMC Genomics. 2005, 6:112.

Neveu, J.; Regeard, C.; Dubow, M.S. Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts. Appl. Microbiol. Biotechnol. 2011, 91: 635–644.

Overbeek, R.; Begley, T.; Butler, R.M.; Choudhuri, J. V; Chuang, H.Y.; Cohoon, M.; de Crécy-Lagard, V.; Diaz, N.; Disz, T.; Edwards, R.; et al. The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res. 2005, 33: 5691–5702.

Pereira, L.B.; Palermo, B.R.Z.; Carlos, C.; Ottoboni, L.M.M. Diversity and antimicrobial activity of bacteria isolated from different Brazilian coral species. FEMS Microbiol. Lett. 2017, 364: 1–8.

Porporato, E.M.D.; Lo Giudice, A.; Michaud, L.; de Domenico, E.; Spanò, N. Diversity and Antibacterial Activity of the Bacterial Communities Associated with Two Mediterranean Sea Pens, Pennatula phosphorea and Pteroeides spinosum (Anthozoa: Octocorallia). Microb. Ecol. 2013, 66: 701–714.

Qin, X.Y.; Yang, K.L.; Li, J.; Wang, C.Y.; Shao, C.L. Phylogenetic diversity and antibacterial activity of culturable Fungi derived from the Zoanthid Palythoa haddoni in the South China Sea. Mar. Biotechnol. 2015, 17: 99–109.

Ram, R. J.; Verberkmoes, N. C.; Thelen, M. P.; Tyson, G. W.; Baker, B. J.; Blake, R. C.; Shah, M.; Hettinch, R. L.; Bandield, J. F. Community proteomics of a natural microbial biofilm. Science. 2005, 308: 1915–1920.

Riesenfeld, C.S.; Schloss, P.D.; Handelsman, J. Metagenomics: Genomic Analysis of Microbial Communities. Annu. Rev. Genet. 2004, 38: 525–552.

Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular cloning: a laboratory manual. Cold Spring Harbor.; 2nd edn. Laboratory Press, 1989.

Schillinger, U.; Lucke, F.K. Antimicrobial activity of Lactobacillus sake isolated from meat. Appl. Environ. Microbiol. 1989, 55: 1901–1906.

Sharma, S.; Khan, F.G.; Qazi, G.N. Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas. Appl. Microbiol. Biotechnol. 2010, 86: 1821–1828.

Sivashankari, S.; Shanmughavel, P. Functional annotation of hypothetical proteins – A review. Bioinformation. 2006, 1: 335-338.

Tasse, L.; Bercovici, J.; Pizzut-serin, S.; Robe, P.; Tap, J.; Klopp, C.; Cantarel, L.B.; Coutinho, P.M.; Henrissat, B.; Leclerc, M.; Dore´, J.; Monsan, P.; Remaud-Simeon, M.; Potocki-Veronese, G. Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes. Genome Res. 2010, 20: 1605–1612.

Tatusov, R.L. The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000, 28: 33–36.

Van Bueren, A.L.; Boraston, A.B. The Structural Basis of ?-Glucan Recognition by a Family 41 Carbohydrate-binding Module from Thermotoga maritima. J. Mol. Biol. 2007, 365: 555–560.

Vihinen, M.; Mantsiila, P. Microbial Amylolytic Enzyme. Crit. Ver. Microbiol. 1989, 24: 329–418.

Wang, D.Z.; Kong, L.F.; Li, Y.Y.; Xie, Z.X. Environmental microbial community proteomics: Status, challenges and perspectives. Int. J. Mol. Sci. 2016, 17: 1–20.

Webster, N.S.; Wilson, K.J.; Blackall, L.L.; Hill, R.T. Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile. Appl. Environ. Microbiol. 2001, 67: 434–444.

Wegley, L.; Edwards, R.; Rodriguez-Brito, B.; Liu, H.; Rohwer, F. Metagenomic analysis of the microbial community associated with the coral Porites astreoides. Environ. Microbiol. 2007, 9: 2707–2719.

Wexler, M.; Bond, P.L.; Richardson, D.J.; Johnston, A.W.B. A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase. Environ. Microbiol. 2005, 7: 1917–1926.




How to Cite

Costa, M. S., Rezende, R. P., Quinto, C. de A., Marques, E. de L. S., Pirovani, C. P., Maciel, B. M., Souza, M. C. B., & Dias, J. C. T. (2020). Outlook of fosmid library from metagenomic of the symbionts associated with coral Siderastrea stellata: structural and functional screening for metabolic and antimicrobial activity / Perspectiva da biblioteca fosmidial a partir da metagenômica de simbiontes associados ao coral Siderastrea stellata: triagem estrutural e funcional da atividade metabólica e antimicrobiana. Brazilian Journal of Development, 6(5), 26371–26392. https://doi.org/10.34117/bjdv6n5-188



Original Papers