26371 Brazilian Journal of Development 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 DOI:10.34117/bjdv6n5-188 Recebimento dos originais: 10/04/2020 Aceitação para publicação: 11/05/2020 Moara Silva Costa Doutora em Biologia e Biotecnologia de Microrganismos Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected] Rachel Passos Rezende Doutora em Ciências Biológicas (Microbiologia) Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected] Cristiane de Araújo Quinto Doutora em Biotecnologia Instituição: Universidade Estadual de Feira de Santana Endereço: Avenida Transnordestina, s/n - Novo Horizonte, Feira de Santana – BA. E-mail: [email protected] Eric de Lima Silva Marques Doutor em Genética e Biologia Molecular Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected] Carlos Priminho Pirovani Doutor em Genética e Biologia Molecular Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected] Braz. J. of Develop., Curitiba, v. 6, n. 5, p.26371-26392, may. 2020. ISSN 2525-8761 26372 Brazilian Journal of Development Bianca Mendes Maciel Doutora em Genética e Biologia Molecular Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected] Maria Clara Bessa Souza Graduanda em Biomedicina Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected] João Carlos Teixeira Dias Doutor em Ciências Biológicas (Microbiologia) Instituição: Universidade Estadual de Santa Cruz. Endereço: Campus Soane Nazaré de Andrade, Rodovia Jorge Amado, km 16, Bairro Salobrinho, Ilhéus-BA. E-mail: [email protected] ABSTRACT 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. Keywords: Biocompound; Cnidaria; Heterologous expression; Proteomics. RESUMO Microrganismos colonizam os corais e produzem compostos com propriedades biológicas interessantes para a biotecnologia. O objetivo do presente estudo foi realizar buscas por biocompostos com potencial industrial através da clonagem de DNA metagenômico de simbiontes do coral S. stellata em vetor fosmídeo. A biblioteca metagenômica foi analisada usando triagem funcional e sequenciamento via Illumina MiSeq. Dos 3648 clones triados foram identificados oito clones proteolíticos e seis amilolíticos. O clone proteolítico P07H3 também apresentou atividade antimicrobiana contra S. aureus, S Enterica PT11, S Enterica PT4 and S Typhi. Não houve identificação de clones positivos para lipase/esterase. Beta-lactamase foi o único subsistema Braz. J. of Develop., Curitiba, v. 6, n. 5, p.26371-26392, may. 2020. ISSN 2525-8761 26373 Brazilian Journal of Development identificado pelo Rapid Annotation using Subsystem Technology (RAST). Entretanto, houve identificações de enzimas que participam de processos metabólicos como o catabolismo de polissacarídeos, óxido-redução, transdução sinal e fosforilação. Quando as proteínas hipotéticas foram re-analisadas elas exibiram um domínio funcional definido. Todas open reading frames (ORFs) identificadas apresentaram baixa identidade com proteínas depositadas no Basic Local Alignment Search Tool protein (BLASTp). O sequenciamento mostra que a maioria dos fragmentos genômicos do clone P07H3 possivelmente consiste em novas proteínas. O filo Proteobacteria foi o com maior predominância entre os fragmentos analisados. Este é o primeiro relato sobre a prospecção de amilases, proteases e antimicrobianos de amostras do coral S. stellata. Portanto, as análises desse estudo ajudam a melhorar o conhecimento a acerca da diversidade metabólica de S. stellata ainda pouco explorada bem como viabiliza no futuro o estudo dos compostos identificados. Palavras-chaves: Biocomposto; Cnidaria; Expressão heteróloga; Proteômica. 1 INTRODUCTION The Zooxanthellae coral Siderastrea stellata (Anthozoa: Scleractinia) is endemic to the Brazilian coast and occurs in shallow water environments and regions susceptible to intense maritime variations (Costa et al., 2001). Moreover, it is reportedly resistant to temperature changes, salinity, and turbidity of water (Leão et al., 2003). This metabolic plasticity together with high biological productivity makes this coral and its symbionts, a promising source in the search for new biomolecules. Only a small portion (0.001%) of microbial diversity in the marine environment is known (Amann et al., 1995). However, technological advances through studies that do not depend on the cultivation of microorganisms have provided knowledge of microbial taxonomic groups (Handelsman et al., 1998). Metagenomic tools with heterologous expression have enabled greater access to metabolic diversity. Through this approach, new metabolic routes, genes, and compounds are being discovered and applied to the understanding of various microbiomes and to the development of numerous biotechnological sectors (Dhanjal and Sharma 2018). Functional and structural metagenomics were used to detect biocompounds for biotechnological purposes. As such, the metagenomic DNA of the coral S. stellata microbiome was cloned in the fosmid vector pCC2FOS using a heterologous host. Samples of S. stellata DNA were tested for the first time to identify antimicrobial compounds and enzymes of economic interest. 2 MATERIAL AND METHODS 2.1 STUDY AREAS AND SAMPLING OF CORAL Siderastrea stellata Coral samples were collected in the Maraú peninsula BA (14º 06' 11" S, 39º 00' 53" W), certified by the National System For The Management of Genetic Heritage and Associated Traditional Knowledge (SisGen A7B90DA). Eight colonies with an approximate diameter of 10 cm were collected randomly in each coral S. stellata. The samples were transported on ice in a Braz. J. of Develop., Curitiba, v. 6, n. 5, p.26371-26392, may. 2020. ISSN 2525-8761 26374 Brazilian Journal of Development polystyrene container for processing to the Microbial Biotechnology Laboratory at the State University of Santa Cruz, Ilhéus, BA. 2.2 CONSTRUCTION OF THE METAGENOMIC LIBRARY OF CORAL MICROBIOME The metagenomic library was constructed according to the standardization of protocols already published in the literature (Wegley et al., 2007; Cheng et al., 2009). For total DNA extraction from the coral, 50g fragments of tissue, mucus, and skeleton were macerated with a mortar and pestle. The resulting macerate was diluted in sodium phosphate buffer (200 mM, pH 7.5) with 1% sodium pyrophosphate (Synth, São Paulo, Brazil) and 1% Tween® 80. The mixture was incubated under constant agitation at 180 g for 24 h at room temperature. The mixture was then centrifuged at 100 g for 5 min. The supernatant was then transferred to a falcon tube and re-subjected to centrifugation of 500 g for 10min. The pellet containing the microbial cells with 3g (wet weight) was used for DNA extraction with the E.Z.N.A.® Soil DNA kit (Omega bio-tek, Inc., Norcross, GA, USA) as per the guidelines of manufacturer. The metagenomic library was constructed using the CopyControl™ HTP Fosmid Library Production kit (Epicentre Biotechnologies, USA). The extracted metagenomic