UNIVERSIDADE ESTADUAL PAULISTA “Júlio de Mesquita Filho” Instituto de Química - Campus Araraquara Marcelo Marucci Pereira Tangerina Fauna Acompanhante: Um Universo Químico a Ser Explorado Tese de Doutorado apresentada como parte dos requisitos para a obtenção do título de Doutor em Química. Orientador: Prof. Dr. Wagner Vilegas Co-Orientador: Prof. Dr. Wagner C. Valenti Araraquara 2016 FICHA CATALOGRÁFICA Tangerina, Marcelo Marucci Pereira T164b By-catch: a chemical universe to be explored = Fauna acompanhante um universo químico a ser explorado / Marcelo Marucci Pereira Tangerina. – Araraquara: [s.n.], 2016 122 p.: il. Tese (doutorado) – Universidade Estadual Paulista, Instituto de Química Orientador: Wagner Vilegas Coorientador: Wagner Cotroni Valenti 1. Fauna marinha. 2. Produtos naturais. 3. Invertebrados marinhos. 4. Bactérias. 5. Actinobactéria. I. Título. Elaboração: Seção Técnica de Aquisição e Tratamento da Informação Biblioteca do Instituto de Química, Unesp, câmpus de Araraquara SÃO PAULO STATE UNIVERSITY “Júlio de Mesquita Filho” Institute of Chemistry - Campus Araraquara Marcelo Marucci Pereira Tangerina By-catch: A Chemical Universe To Be Explored Doctoral Thesis presented as part of the requirements for obtaining the title of Doctor in Chemistry. Supervisor: Prof. Dr. Wagner Vilegas Co-Supervisor: Prof. Dr. Wagner C. Valenti Araraquara 2016 Dados Curriculares Dados Pessoais Nome: Marcelo Marucci Pereira Tangerina Endereço Profissional: 1) Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, Instituto de Química, Departamento de Química Orgânica, Laboratório de Fitoquímica Rua Prof. Francisco Degni, 55. Jd. Quitandinha, CEP: 14800-900 – Araraquara – SP. Telefone: 16 3301-9792 2) Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, Instituto de Biociências, Laboratório de Bioprospecção de Produtos Naturais Praça Infante Dom Henrique, s/n. Parque Bitaru CEP: 11330-900 – São Vicente – SP. Telefone: 13 3569-7169 E-mail: marcelomptang@gmail.com; marcelomptang@hotmail.com Formação Acadêmica Graduação Bacharelado Instituição: Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, Instituto de Química, Campus Araraquara Local: Araraquara - SP Curso: Bacharelado em Química com Atribuições Tecnológicas Período: 2006-2009 Trabalho Orientado em Química – Departamento de Química Inorgânica: - Investigação de Compostos de Ni(II) Coordenado a Diaminas e Pseudohaletos – Bolsista CNPq - Investigação de compostos de Ni(II) coordenado a azida e aminoalcoois como precursores de filmes metálicos – Bolsista FAPESP Orientadora: Prof.ª Dr.ª Vânia Martins Nogueira Pós Graduação Mestrado Instituição: Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, Instituto de Química, Departamento de Química Orgânica, Campus Araraquara Local: Araraquara - SP Período: 2010-2011 Bolsista: FAPESP – Processo no. 2010/04077-0 Título da Dissertação: Extratos Padronizados para o Tratamento de Doenças Crônicas: Machaerium hirtum Orientador: Prof. Dr. Wagner Vilegas Co-Orientadora: Prof.ª Dr.ª Miriam Sannomiya Doutorado Instituição: Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, Instituto de Química, Departamento de Química Orgânica, Campus Araraquara Local: Araraquara – SP/ São Vicente – SP Período: 2012-2016 Bolsista: FAPESP – Processo no. 2011/23159-0 Título da Tese: Fauna Acompanhante: Um Universo Químico a Ser Explorado Orientador: Prof. Dr. Wagner Vilegas Co-Orientador: Prof.ª Dr.ª Wagner C. Valenti Estágio em Instituição de Ensino no Exterior: 1) Instituição: University of Regensburg – UR Local: Regensburg, Alemanha Período: Agosto/2013 – Dezembro/2013 Bolsista: CAPES Projeto: Diversidade e ecologia de caranguejos de manguezal do sudeste do Brasil exemplificado pelas comparações genéticas, morfológicas, etológicas, fisiológicas e ecotoxicológicas de duas espécies intimamente relacionadas de caranguejos violinistas. Orientador no Exterior: Prof. Dr. Christoph D. Schubart Orientador no Brasil: Prof.a Dr.a Tânia Márcia Costa 2) Instituição: University of Prince Edward Island – UPEI Local: Charlottetown, PE, Canadá Período: Julho/2014 – Março/2015 Bolsista: FAPESP (BEPE – Processo no. 2014/08787-2) Projeto: Fauna Acompanhante: Um universo Químico a Ser Explorado Trabalho realizado: Isolamento de bactérias marinhas e bioprospecção das mesmas Orientador no Exterior: Prof. Dr. Russell G. Kerr Orientador no Brasil: Prof. Dr. Wagner Vilegas “Aos meus pais que sempre me apoiaram em todas as decisões que tomei até aqui e à toda minha família, dedico este trabalho.” Agradecimentos Agradeço aos meus pais, minha avó e meu irmão por ser quem são, pessoas excepcionais que sempre me apoiaram, compartilharam minhas alegrias, minhas angústias e se fizeram presentes por todo caminho que trilhei. Ao meu orientador, Wagner Vilegas, que formou o profissional que sou hoje. Sempre será meu orientador, o qual respeito muito, mas considero também um grande amigo que ganhei durante minha pós-graduação e hoje tenho em grande estima. Aos meus colegas e amigos do laboratório Claudinha, Douglas, Polly, Mayara, Marcelo, Julia, Lucas, Luiza, Nis, pela ajuda no laboratório, conversas informais, desabafos, cafés e todas as confraternizações. Aos meus grandes amigos que conheço desde minha graduação Jerso, Miguel, Glauco, Mari e Fabi pelas risadas e amizade verdadeira. Ao bando de biólogos que conheci durante o período no Campus de São Vicente da UNESP, Pri, Kiwi, Tixa, Dona, Shan, Ana, Cala, Bartira, Vem, Fer e a tantos outros aqui não citados. Aos professores da UNESP de São Vicente, Wagner C. Valenti e Tânia Márcia Costa e à professora do Instituto de Química de Araraquara, Lourdes Campaner pelas oportunidades e ótimo convívio. Ao Prof. Russell Kerr pela oportunidade de estágio em seu laboratório na University of Prince Edward Island – Canada e a todos que me ajudaram, em especial Hebelin, Brad e Fabrice. À Rede SAO-MAR/CNPq. À Capes pelo financiamento do estágio no exterior na Alemanha. À FAPESP pelas bolsas concedidas no país (Processo no. 2011/23159-0) e no exterior (BEPE - Processo no. 2014/08787-2). “It must be a strange world not being a scientist, going through life not knowing – or maybe not caring about where the air came from, where the stars at night came from or how far they are from us. I Want To Know.” - Michio Kaku Resumo Expandido O desperdício de recursos provenientes da biodiversidade é enorme no Brasil e no mundo. A grande variedade de organismos marinhos (algas, moluscos, esponjas, corais, etc) que são pescados em conjunto com peixes e camarões, chamada de fauna acompanhante, é completamente ignorada como fonte de novas substâncias. Devido ao baixo valor agregado, os pescadores os desprezam, pois não são comercializáveis como alimento. A pesca de camarões é uma das mais impactantes aos ecossistemas marinhos: o uso de sistemas de arrasto do fundo marinho provoca a destruição de todo a região onde ele é empregado, capturando até 21 kg de fauna acompanhante/kg de camarão. Diversas substâncias encontradas em organismos marinhos são extensamente aplicadas em diversas áreas para a melhoria da qualidade de vida da humanidade, incluindo a área de alimentos, de geração de energia e medicamentos. Tendo em vista esse quadro, este projeto visou o estudo químico de espécies da fauna acompanhante da pesca do camarão no litoral paulista, visando obter substâncias de potencial interesse econômico, que possam aumentar o valor agregado desse material desperdiçado. Para o desenvolvimento do trabalho foram realizados três arrastos de fundo nas regiões de Ubatuba, Guarujá e Itanhaém, no estado de São Paulo, juntamente aos pescadores camaroeiros locais. O material coletado foi levado ao Campus de São Vicente da UNESP, onde foi triado e as espécies coletadas devidamente identificadas. Um total de 23 taxa de invertebrados (entre crustáceos, moluscos, cnidários e equinodermos), 07 elasmobrânquios (peixes cartilaginosos) e 64 peixes ósseos foram identificados, sendo os invertebrados identificados pela Prof.a Dr.a Tânia M. Costa, e os vertebrados pelos Prof. Dr. Otto Gadig (elasmobrânquios) e Prof. Dr. Teodoro Vasquez (peixes ósseos), docentes da UNESP/CLP. Esta parte do trabalho gerou o artigo intitulado “Biological and preliminary chemical characterization of the by-catch of the shrimp fishery from the São Paulo State coast, Brazil”, submetido ao periódico Latin American Journal of Aquatic Research Os peixes ósseos e os elasmobrânquios, por não ser foco do trabalho, não foram avaliados quimicamente. Quanto aos invertebrados, apesar de uma grande variedade de espécies, a primeira dificuldade encontrada foi a quantidade de indivíduos coletados por espécie. Assim, a maioria dos invertebrados coletados não apresentou massa suficiente para estudos aprofundados de composição química. Dentre as espécies de invertebrados que apresentaram uma maior quantidade de material, foram estudadas as águas-vivas Olindia sambaquiensis, Chrysaora lactea e Chiropsalmus quadrumanus, os gastrópodes Olivancillaria urceus e Buccinanops cochlidium, os cefalópodes Lolliguncula brevis e Dorytheutis plei e o equinodermo Luidia senegalensis. Entretanto, excetuando a espécie de equinodermo, as demais apresentaram apenas metabólitos primários nos estudos realizados. O estudo de L. senegalensis foi possível por meio de espectrometria de massas uma vez que não foi capturado um número elevado de indivíduos. Para tanto, foi utilizada uma combinação de extração em fase sólida (SPE) seguida de cromatografia líquida de ultra eficiência acoplada a um espectrômetro de massas com ionização por electrospray e analisador íon-trap linear (UPLC-ESI-IT-MSn). Foi também realizada análise por injeção direta utilizando a mesma fonte de ionização e mesmo analisador, com fragmentação sequencial dos íons detectados (FIA-ESI-IT-MSn). A espécie apresentou asterosaponinas, as quais são esteroides glicosilados sulfatados, contendo cinco ou seis unidades de açúcar, além de poliidroxiesteroides. Tais resultados evidenciaram a presença de importantes substâncias potencialmente bioativas em invertebrados provenientes da fauna acompanhante da pesca do camarão utilizando um método rápido e eficiente. Esta etapa resultou no artigo “Chemical profile of the sulphated saponins from the starfish Luidia senegalensis collected as by-catch fauna in Brazilian coast”, submetido ao periódico Journal of the Brazilian Chemical Society. Devido à baixa abundância de produtos naturais detectados nos invertebrados provenientes da fauna acompanhante, a busca por novas substâncias foi ainda estendida às espécies microbianas associadas ao material de estudo. Assim, foram também estudadas as bactérias marinhas associadas aos invertebrados coletados e ao sedimento marinho do mesmo local de coleta quanto ao seu potencial na produção de substâncias. Foi explorado o potencial biotecnológico das bactérias cultiváveis de duas espécies de invertebrados da fauna acompanhante, o gastrópode Olivancillaria urceus e a estrela-do-mar L. senegalensis. Foi também estudada uma amostra de sedimento marinho proveniente da mesma área de coleta dos invertebrados. Utilizando múltiplas técnicas de isolamento foram obtidos 134 isolados bacterianos dos invertebrados e do sedimento. O sequenciamento parcial da subunidade do gene rRNA (16S) revelou que os isolados pertencem as filos Proteobacteria, Firmicutes e Actinobacteria, distribuídos em 28 gêneros. Diversos gêneros conhecidos por sua capacidade de produção de substâncias bioativas (Micromonospora, Streptomyces, Serinicoccus e Verrucosispora) foram obtidos das amostras estudadas. A fim de investigar as bactérias quanto a sua capacidade de produção de metabólitos bioativos, os isolados foram cultivados e o caldo de fermentação analisado por cromatografia líquida de ultra eficiência acoplada a detector de arranjo de fotodiodos, detector evaporativo de espalhamento de luz e espectrômetro de massas de alta resolução com ionização por electrospray e analisador orbitrap (UPLC-PDA-ELSD-HRMS) e testados por sua atividade antimicrobiana. Por fim, quatro cepas apresentaram atividade antimicrobiana contra Staphylococcus aureus resistente à meticilina (MRSA) e Staphylococcus warneri. Esta etapa resultou no artigo intitulado “Cultivable bacterial communities of marine sediment and invertebrates from the underexplored Ubatuba region of Brazil”, submetido ao periódico Archives of Microbiology. A produção de substâncias por bactérias é altamente dependente do meio de cultura no qual estas são cultivadas. Ademais, diferentes cepas da mesma espécie podem apresentar diferente produção de metabólitos. A fim de avaliar tais variáveis foi realizado o cultivo em pequena escala em nove meios de cultura diferentes de três cepas da espécie Verrucosispora maris, provenientes do sedimento marinho e de L. senegalensis. Devido ao grande número de amostras obtidas, foi realizado um estudo metabolômico utilizando LC-HRMS e análise dos componentes principais (PCA), onde foi investigada a produção de abyssomicinas (marcadores químicos do gênero que apresentam propriedades anticancerígenas) e outros metabólitos secundários. Foi possível detectar a produção de abyssomicinas somente por uma das cepas avaliadas (RKMT_111) e o estudo da influência da composição do meio de cultura na produção de substâncias revelou que tais metabólitos só foram produzidos em um dos meios de cultura (BFM-11m). Apesar das três cepas pertencerem à mesma espécie e serem provenientes do mesmo local de coleta, é notável que todas apresentaram diferente capacidade de produção de metabólitos secundários. Tais resultados evidenciam a importância de uma triagem prévia dentre cepas de uma determinada espécie e da otimização da composição do meio de cultura a ser utilizado antes de fermentações em larga escala para a produção e posterior isolamento de substâncias provenientes de bactérias. Esta etapa do trabalho resultou no artigo intitulado “Survey of the secondary metabolome of the marine actinomycete Verrucosispora sp”, a ser submetido no periódico Molecules. Além da produção de metabólitos secundários as bactérias podem ainda realizar biotransformações, ou seja, modificações na estrutura de substâncias modificando suas propriedades físico-químicas. A fim de detectar tais transformações, foram estudados os produtos da fermentação de Erythrobacter vulgaris, isolado de sedimento marinho. Para tanto, o isolado foi fermentado em escala ampliada, o caldo de fermentação extraído e as substâncias isoladas por meio de cromatografia líquida preparativa utilizando detector de massas (HPLC-MS). As frações obtidas foram analisadas por UPLC-PDA-ELSD-HRMS e Ressonância Magnética Nuclear (RMN) e foram identificados dois novos derivados do ácido cólico, o ácido 3-acetil-glicocólico e o ácido 3-acetil-glicodesoxicólico. Sugere-se que as duas novas substâncias isoladas foram produzidas por meio da biotransformação dos ácidos glicocólico e glicodesoxicólico, respectivamente, previamente presentes no meio de cultivo. Este é o primeiro registro dos compostos identificados bem como o primeiro estudo em que foi observada uma acilação realizada por um isolado marinho de Erythrobacter vulgaris. Estes resultados geraram o artigo intitulado “New glycocholic and glycodeoxycholic acid derivatives produced by biotransformation in Erythrobacter vulgaris marine isolate”, a ser submetido no periódico Journal of Biotechnology. A busca por novos compostos que sirvam de inspiração para o desenvolvimento de substâncias úteis para a humanidade é cada vez mais difícil e novas fontes devem ser buscadas a cada estudo. A fauna acompanhante mostrou ser uma fonte rica e ainda inexplorada na busca de bactérias marinhas com diversas aplicações biotecnológicas, ainda a serem exploradas. Quanto à composição química dos invertebrados estudados, a fauna acompanhante mostrou-se pouco promissora devido à baixa abundância de cada espécie e à grande captura de animais pelágicos, os quais apresentam outros mecanismos de defesa que não sejam a produção de substâncias químicas. Resumo A fauna acompanhante da pesca do camarão inclui uma série de invertebrados marinhos que são descartados por não ter valor comercial. A fim de tentar acrescentar algum valor a este material, foi analisada a composição química da estrela-do-mar Luidia senegalensis coletada na costa brasileira como consequência da aplicação da pesca de arrasto. A fim de avaliar sua composição química, foi utilizada uma combinação de extração em fase sólida (SPE) seguida de cromatografia líquida de ultra eficiência acoplada a espectrômetro de massas equipado com fonte de ionização por eletrosptray e analisador ion-trap linear (UPLC- ESI-IT-MSn). Luidia senegalensis contém asterosaponinas, que são esteroides glicosilados sulfatados contendo cinco e seis unidades de açúcar, além de poliidroxiesteroides. Este estudo mostrou a presença de compostos importantes e potencialmente bioativos em invertebrados associados à fauna acompanhante da pesca do camarão, usando um método rápido e eficiente. Normalmente descartada, a fauna acompanhante contém muitos invertebrados que podem hospedar uma grande variedade de gêneros de bactérias, algumas das quais com potencial de produzir produtos naturais bioativos com aplicações biotecnológicas. Assim, para utilizar um material normalmente descartado, foi explorado o potencial biotecnológico de bactérias cultiváveis de duas espécies de invertebrados abundantes na fauna acompanhante, o gastrópode Olivancillaria urceus e a estrela-do-mar Luidia senegalensis. Uma amostra de sedimento da mesma área de coleta também foi investigado. Utilizando múltiplas abordagens de isolamento 134 isolados foram obtidos a partir dos invertebrados e do sedimento. Sequenciamento parcial da subunidade de rRNA (16S) revelou que os isolados pertenciam aos filos Proteobacteria, Firmicutes e Actinobacteria, distribuídos em 28 gêneros. Vários gêneros conhecidos pela sua capacidade de produzir produtos naturais bioativos (Micromonospora, Streptomyces, Serinicoccus e Verrucosispora) foram obtidos a partir das amostras estudadas. Para avaliar as bactérias isoladas quanto à sua capacidade para produzir metabólitos bioativos todas as cepas foram fermentadas e os extratos de fermentação analisados por LC-HRMS e testados em ensaio de atividade antimicrobiana. Quatro cepas apresentaram atividade antimicrobiana contra Staphylococcus aureus resistente à meticilina (MRSA) e Staphylococcus warneri. A produção de metabólitos secundários por bactérias isoladas da fauna acompanhante também foi avaliada por uma abordagem metabolômica utilizando LC-HRMS, onde foi avaliado como as diferenças na composição dos meios de cultura podem alterar a produção de substâncias. Utilizou-se a metabolômica como uma ferramenta para investigar a produção de abyssomicinas, um agente anticâncer, e outros metabólitos secundários em três cepas do actinomiceto raro Verrucosispora maris, isoladas a partir de uma amostra de sedimento e associadas à estrela-do-mar Luidia senegalensis de Ubatuba - SP, Brasil. Nove composições diferentes de meios de cultura foram avaliadas e verificou-se que, dentre todas as cepas, somente RKMT_111 foi capaz de produzir abyssomicinas. O estudo da composição do meio de cultura revelou que a produção de abyssomicinas só foi possível em BFM-11m. Embora as três cepas pertençam à mesma espécie e são provenientes da mesma localização, é notável que cada isolado apresentou diferente capacidade de produção de metabólitos secundários. Os produtos de fermentação de Erythrobacter vulgaris foram avaliados utilizando técnicas de HPLC preparativo, LC-HRMS e RMN. A cepa foi isolada pelo método dry-stamp de uma amostra de sedimento marinho da costa de Ubatuba-SP, Brasil. Depois de sequenciamento completo do rRNA (16S) e identificação, o isolado foi fermentado em larga escala, seu caldo de fermentação extraído por solvente e os compostos purificados por HPLC- MS. Análise de LC-HRMS e RMN dos compostos isolados levou à identificação de dois novos derivados do ácido cólico, ácido 3-acetil-glicocólico e o ácido 3-acetil- glicodesoxicólico. As substâncias obtidas podem ter sido produzidas por biotransformação do ácido glicocólico e ácido desoxicólico, respectivamente, já presentes no meio de cultivo. Este é o primeiro relato de tais compostos e também a primeira observação de uma acilação realizada por um isolado marinho de Erythrobacter vulgaris. Palavras-chave: Fauna acompanhante; produtos naturais marinhos; invertebrados; bactérias Abstract The by-catch fauna of the shrimp fishery includes a number of marine invertebrates that are discarded because they do not have commercial value. In order to try to add some value to these materials, we analyzed the chemical composition of the starfish Luidia senegalensis collected in the Brazilian coast as a consequence of the trawling fishery method. In order to access their chemical composition, we used a combination of solid phase extraction (SPE) followed by ultra performance liquid chromatography coupled to electrospray ionization ion trap tandem mass spectrometry (UPLC-ESI-IT-MSn). Luidia senegalensis contains asterosaponins, which are sulphated glycosilated steroids, containing five and six sugar moieties, in addition to polyhydroxysteroids. This study helped us to support the presence of important and potentially bioactive compounds in invertebrates associated to the by-catch fauna of the shrimp fishery, using a fast and efficient method. Typically discarded, by-catch contains many invertebrates that may host a great variety of bacterial genera, some of which may produce bioactive natural products with biotechnological applications. Therefore, to utilize by-catch that is usually discarded we explored the biotechnological potential of culturable bacteria of two abundant by-catch invertebrate species, the snail Olivancillaria urceus and the sea star Luidia senegalensis. Sediment from the collection area was also investigated. Utilizing multiple isolation approaches 134 isolates were obtained from the invertebrates and sediment. Small subunit rRNA (16S) gene sequencing revealed that the isolates belonged to Proteobacteria, Firmicutes and Actinobacteria phyla and were distributed among 28 genera. Several genera known for their capacity to produce bioactive natural products (Micromonospora, Streptomyces, Serinicoccus and Verrucosispora) were retrieved from the invertebrate samples. To query the bacterial isolates for their ability to produce bioactive metabolites all strains were fermented and fermentation extracts profiled by LC-HRMS and tested for antimicrobial activity. Four strains exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus warneri. The production of secondary metabolites was assessed using a LC-HRMS-based metabolomics approach, where it was evaluated how differences in media composition can alter the production of chemical compounds. We used metabolomics as a tool to investigate the production of abyssomicins, an anticancer agent, and other secondary metabolites in three strains of the rare actinomycete Verrucosispora maris, all marine isolates from a sediment sample and associated to a starfish from the species Luidia senegalensis of Ubatuba – SP, Brazil. Nine different media compositions were evaluated and it was found that, among all strains, only RKMT_111 was capable of producing abyssomicins. The media composition study revealed that the production of abyssomicins was only achievable in BFM-11m. Although the three strains belong to the same species and the same location, it is worthwhile noticing that each isolate showed different capability for production of secondary metabolites. The products of fermentation of Erythrobacter vulgaris were evaluated using preparative HPLC, LC-HRMS and NMR techniques. Bacterial strain was isolated by dry- stamp method from a marine sediment sample from the coast of Ubatuba-SP, Brazil. After fully 16S rDNA sequence and identification, the marine isolate was fermented in large-scale, extracted and the compounds purified through HPLC-MS. Analysis of LC-HRMS and NMR of the isolated compounds led to the identification of two new cholic acid derivatives, 3- acetyl-glycocholic acid and 3-acetyl-glycodeoxycholic acid. Both new compounds may have been produced by the biotransformation of glycocholic acid and deoxycholic acid, respectively, already present in the cultivation medium. This is the first report of such compounds and also the first time an acylation has been observed for an Erythrobacter vulgaris marine isolate. Keywords: By-catch; marine natural products; invertebrates; bacteria Lista de Figuras Figure 1. Full scan DFI-ESI-IT-MSn (Negative Ionization) mass spectrum of Luidia senegalensis showing the peaks corresponding to the saponins ............................................... 48 Figure 2. UPLC-ESI-IT-MS analysis of the saponins present in Luidia senegalensis. Base Peak Ion -BPI (above) and extracted chromatograms of the ions 1: m/z 1405; 2: m/z 1389; 4: m/z 1239; 3 and 5: 1227; 6: m/z 529; 7: m/z 547 ...................................................................... 49 Figure 3. Aglycones of the steroidal saponins detected in Luidia senegalensis. R corresponds to the sugar sequence described in Table 2 .............................................................................. 50 Figure 4. Mass spectrum obtained after UPLC-ESI-IT-MSn experiment using the precursor ion ............................................................................................................................................. 52 Figure 5. Phylogenetic analysis of Proteobacteria isolates obtained from Brazilian sediment and the invertebrates Luidia senegalensis and Olivancillaria urceus. Prior to tree construction sequences sharing >99% sequence identity were grouped into OTUs and a single representative of each OTU was used in the phylogenetic analysis. The sources of isolates belonging to an OTU are indicated by symbols which follow the strain number: wet sediment - n; dry sediment - t; Luidia senegalensis - p; Olivancillaria urceus - l. Neighbor-joining tree constructed using MEGA 6. The analysis considered 577 nucleotides. Bootstrap values greater than 50% are shown at the nodes and are based on 1000 iterations. The scale bar represents the number of base substitutions per site ................................................................. 58 Figure 6. Phylogenetic analysis of Firmicutes isolates obtained from Brazilian sediment and the invertebrates Luidia senegalensis and Olivancillaria urceus. Prior to tree construction sequences sharing >99% sequence identity were grouped into OTUs and a single representative of each OTU was used in the phylogenetic analysis. The sources of isolates belonging to an OTU are indicated by symbols which follow the strain number: wet sediment - n; dry sediment - t; Luidia senegalensis - p; Olivancillaria urceus - l. Neighbor-joining tree constructed using MEGA 6. The analysis considered 573 nucleotides. Bootstrap values greater than 50% are shown at the nodes and are based on 1000 iterations. The scale bar represents the number of base substitutions per site ................................................................. 59 Figure 7. Phylogenetic analysis of Actinobacteria isolates obtained from Brazilian sediment and the invertebrates Luidia senegalensis and Olivancillaria urceus. Prior to tree construction sequences sharing >99% sequence identity were grouped into OTUs and a single representative of each OTU was used in the phylogenetic analysis. The sources of isolates belonging to an OTU are indicated by symbols which follow the strain number: wet sediment - n; dry sediment - t; Luidia senegalensis - p; Olivancillaria urceus - l. Neighbor-joining tree constructed using MEGA 6. The analysis considered 621 nucleotides. Bootstrap values greater than 50% are shown at the nodes and are based on 1000 iterations. The scale bar represents the number of base substitutions per site ................................................................. 60 Figure 8. Effect of media composition in the production of secondary metabolites of three strains of Verrucosispora maris. Principal component analysis (PCA) – Scores plot (left) and loadings plot (right), PC-1 versus PC-2. (A) Strain RKMT_073; (B) Strain RKMT_111; (C) Strain RKMT_176; (D) All three strains analyzed together ..................................................... 68 Figure 9. Chemical barcoding and cluster analysis of the three strains of Verrucosispora maris in all media tested ........................................................................................................... 74 Figure 10. Phylogenetic analysis of RKMT_070 isolate obtained from Brazilian sediment and reference strains from Genbank. Neighbor-joining tree constructed using MEGA 6. The analysis considered 1336 nucleotides. Bootstrap values greater than 50% are shown at the nodes and are based on 1000 iterations. The scale bar represents the number of base substitutions per site ................................................................................................................. 75 Figure 11. Structure of compounds isolated from Erythrobacter vulgaris fermentation ........ 78 Figure S1. Chromatographic profile of the extract from Streptomyces sp. strain RKMT_071. ELSD, HRMS and PDA detectors. Below, extracted chromatograms of the ions of m/z 479.29080 [M+H]+, 1111.64453 [M+H]+, 1147.64099 [M+Na]+ and 560.31848 [M+H]+. .. 101 Figure S2. UV profile of the peak at retention time of 4.54 min in the PDA detector from the analysis of the extract from RKMT_071 strain ...................................................................... 101 Figure S3. Chromatographic profile of the extract from Micromonospora sp. strain RKMT_160. ELSD, PDA and HRMS detectors. Below, extracted chromatograms of the ions of m/z 261.12335 [M+H]+ and 284.13940 [M+H]+ ................................................................ 102 Figure S4. Chromatographic profile of the extract from Bacillus sp. strain RKMT_178. ELSD, PDA and HRMS detectors. Below, extracted chromatograms of the ions of m/z 345.18419 [M+H]+ and 389.19112 [M+H]+ ........................................................................... 102 Figure S5. Chromatographic profile of the extract from Halobacillus sp. strain RKMT_184. ELSD, PDA and HRMS detectors .......................................................................................... 103 Figure S6. Chromatographic profile of the extract from Halobacillus sp. strain RKMT_184. ELSD and HRMS detectors. Below, extracted chromatograms of the ions of m/z 316.12922 [M+H]+, 351.12129 [M+H]+, 325.12518 [M+H]+, 217.09728 [M+H]+, 349.17912 [M+H]+ and 375.17545 [M+H]+ ................................................................................................................. 103 Figure S7. Extracted chromatograms in SIM mode for the ion [M+H]+ of m/z 231.1016 from RKMT_111 (top) and RKMT_176 (bottom) in BFM-11m corresponding to the compound Methyl 3-methoxy-5-methyl-naphthalene-1-carboxylate ....................................................... 104 Figure S8. Extracted chromatograms in SIM mode for the ion [M+H]+ of m/z 257.1172 corresponding to the compound kurasoin A. A – RKMT_111 in BFM-1m; B – RKMT_176 in BFM-1m; C – RKMT_111 in BFM-11m; D – RKMT_176 in BFM-11m. ........................... 104 Figure S9. Extracted chromatograms in SIM mode for the ion [M+H]+ of m/z 247.1082 from RKMT_111 (top) and RKMT_176 (bottom) in BFM-11m corresponding to the compound anthramycin ............................................................................................................................ 105 Figure S10. Extracted chromatograms in SIM mode for the ion [M+H]+ of m/z 223.0964 from RKMT_111 (top) and RKMT_176 (bottom) in BFM-11m corresponding to the compound talomone ................................................................................................................................. 105 Figure S11. Extracted chromatograms in SIM mode for the ion [M+H]+ of m/z 219.1016 from RKMT_111 (top) and RKMT_176 (bottom) in BFM-11m corresponding to the compound 4H-1,3-Benzodioxin-4-one, 2,2-dimethyl-5-(2-propen-1-yl)- ............................................... 106 Figure S12. Extracted chromatograms in SIM mode for the ion [M+H]+ of m/z 293.1383 from RKMT_111 (top) and RKMT_176 (bottom) in BFM-11m corresponding to the compound 4H-1-Benzopyran-4-one, 2-butyl-8-hydroxy-5,7-dimethoxy-3-methyl- ............................... 106 Figure S13. Extracted chromatograms in SIM mode for the ion [M+H]+ of m/z 521.3475 from RKMT_111 (top) and RKMT_176 (bottom) in BFM-4m corresponding to the compound butyrolactol ............................................................................................................................. 107 Figure S14. Comparison of the composition of all fractions obtained by extraction with ethyl acetate and HP-20 of the broth from cultivation of RKMT_070 in tubes and baffled flasks. Left: ELSD detector; Center: PDA detector; Right: HRMS detector. A to D: cultivation in baffled flasks, ethyl-acetate, HP-20 H2O, HP-20 MeOH/H2O, HP-20 MeOH, respectively. E to H: cultivation in tubes, ethyl-acetate, HP-20 H2O, HP-20 MeOH/H2O, HP-20 MeOH, respectively. ............................................................................................................................ 117 Figure S15. Chromatogram of preparative HPLC-MS of fraction HP-20 MeOH. Sunfire C18 column (5 µm, 250 × 10 mm, 110 Å, Waters®). Method described in Isolation section. Numbers in the chromatogram correspond to the method fractions were collected .............. 117 Figure S16. Compounds detected in the medium blank analysis by LC-HRMS. A: Base Peak chromatogram of the medium blank. Extracted ion chromatograms of the ions B: m/z 466.4; C: m/z 407.3; D: m/z 409.3; E: m/z 450.3 ............................................................................... 118 Figure S17. Extraction of the ions of m/z 492.3 (B) and m/z 508.3 (C) from analysis of the medium blank (A), confirming the absence of the isolated compounds in the medium composition ............................................................................................................................ 118 Figure S18. 1H-NMR spectrum of Fraction 07 (14.0 T, MeOD, ppm) ................................. 119 Figure S19. HMQC contour map of Fraction 07 (14.0 T, MeOD, ppm) ............................... 119 Figure S20. HMBC contour map of Fraction 07 (14.0 T, MeOD, ppm) ............................... 120 Figure S21. 1H-NMR spectrum of Fraction 11 (14.0 T, MeOD, ppm) ................................. 120 Figure S22. HMQC contour map of Fraction 11 (14.0 T, MeOD, ppm) ............................... 121 Figure S23. HMBC contour map of Fraction 11 (14.0 T, MeOD, ppm) ............................... 121 Lista de Tabelas Table 1. Species belonging to Verrucosispora genus, location and source of first report ...... 28 Table 2. Proposed saponins present in Luidia senegalensis detected by ESI-IT-MSn (1), UPLC-ESI-IT-MS and UPLC-ESI-IT-MSn (2) in negative ion mode ....................................... 50 Table 3. Summary of isolates obtained from different samples using different pretreatments and isolation media. Numbers in represent “number of isolates/number of genera” ............... 57 Table 4. Taxonomic affiliation of representative OTUs of bacteria isolated from Brazilian sediment and the invertebrates L. senegalensis and O. urceus. Type strains for comparisons were identified from BlastN searches of the GenBank 16S rRNA gene sequence database. Source: O - Snail; L - Sea Star; D - Dry Sediment; W - Wet Sediment. Pretreatment: U - Untreated; DS - Dry-stamp; H - Heat; P - Phenol; DDC2 - Dispersial and Differential Centrifugation ........................................................................................................................... 61 Table 5. Antimicrobial activity of fermentation extracts derived from four strains. No activity was observed against the other four pathogens (E. faecium, P. aeruginosa, P. vulgaris and C. albicans). No antimicrobial activity was observed in extracts from other strains examined ... 63 Table 6. Summary of metabolites observed in UPLC-HRMS analyses of fermentation extracts from four strains. To identify metabolites detected in these analyses, the pseudomolecular (PM) ion m/z was used to search the Antibase 2014 database using a 5 ppm window above and below the observed molecular weight (MW). A 10 ppm search window was used for pseudomolecular ions with m/z >1000. Compounds with no matches in Antibase were considered putatively novel .............................................................................. 66 Table 7. Chromatographic data of the fractionation and analysis by LC-HRMS of the fractions .................................................................................................................................... 77 Table 8. Chemical shifts of glycocholic, glycodeoxycholic (IJARE et al., 2005), 3-acetyl- glycocholic and 3-acetyl-glycodeoxycholic acids (MeOD) ..................................................... 79 Table S1. Detected ions produced by LC-HRMS from the three strains of Verrucosispora maris isolated and media where they were detected. Compounds identified by comparison to (1) Antibase and (2) Scifinder database ................................................................................. 108 Sumário 1. Introduction ........................................................................................................................ 23 1.1. The by-catch fauna and marine invertebrates ............................................................ 23 1.2. Marine bacteria and its biotechnological potential .................................................... 25 1.3. Metabolomics and the chemical study of marine bacteria ......................................... 26 1.4. Proteobacteria and biotransformations ...................................................................... 29 2. General and Specific Goals ............................................................................................ 31 3. Materials and Methods .................................................................................................. 32 3.1. Mass spectrometry study of the starfish Luidia senegalensis .................................... 32 3.1.1. Chemicals and Materials ........................................................................................ 32 3.1.2. Animal material, extraction and fractionation ....................................................... 32 3.1.3. ESI-MS n analysis .................................................................................................... 33 3.1.4. UPLC-ESI-IT-MS analysis ..................................................................................... 34 3.2. Marine bacteria .......................................................................................................... 34 3.2.1. Cultivable bacterial communities of marine sediment and invertebrates .............. 34 3.2.1.1. Sample collection ............................................................................................ 34 3.2.1.2. Methods for the isolation of bacteria ........................................................... 35 3.2.1.3. Culture media for bacterial isolation ........................................................... 36 3.2.1.4. Bacteria identification ................................................................................. 37 3.2.1.5. Phylogenetic analysis of cultured bacteria .................................................. 38 3.2.1.6. Bacteria cultivation and extraction .............................................................. 38 3.2.1.7. Antimicrobial assays ................................................................................... 39 3.2.1.8. UPLC-HRMS analysis ................................................................................ 39 3.2.2. Metabolomic study of the marine actinomycete Verrucosispora sp. .................. 40 3.2.2.1. Bacteria isolation and identification ............................................................ 40 3.2.2.2. Bacteria fermentation .................................................................................. 40 3.2.2.3. Extraction and LC-HRMS analysis ............................................................. 41 3.2.2.4. Preprocessing and statistical analysis .......................................................... 42 3.2.2.5. Identification of compounds ........................................................................ 43 3.2.3. Biotransformations in bacterial isolates ............................................................ 43 3.2.3.1. Bacteria isolation and identification ............................................................ 43 3.2.3.2. Fermentation, extraction and LC-HRMS analysis ...................................... 44 3.2.3.3. Isolation and identification of compounds .................................................. 45 4. Results .............................................................................................................................. 46 4.1. Mass spectrometry study of the starfish Luidia senegalensis…..………………...….46 4.1.1. SPE fractionation of the crude extract ................................................................... 47 4.1.2. DFI-ESI-IT-MS n analyses ....................................................................................... 47 4.1.3. UPLC-ESI-IT-MS analysis ..................................................................................... 48 4.2. Marine bacteria .......................................................................................................... 54 4.2.1. Cultivable bacterial communities of marine sediment and invertebrates .............. 54 4.2.1.1. Bacteria isolation and identification ............................................................ 54 4.2.1.2. Phylogenetic analysis .................................................................................. 58 4.2.1.3. Antimicrobial assay ..................................................................................... 63 4.2.1.4. Chemical analysis of bioactive extracts ...................................................... 63 4.2.2. Metabolomic study of the marine actinomycete Verrucosispora sp. ...................... 66 4.2.2.1. Identification of bacterial strains ................................................................. 66 4.2.2.2. Bacteria fermentation and metabolite production evaluation ..................... 67 4.2.2.3. Metabolite production of the isolated strains .............................................. 67 4.2.2.4. Compilation of the data ............................................................................... 68 4.2.2.5. RKMT_073 metabolites production ............................................................ 69 4.2.2.6. RKMT_111 metabolites production ............................................................ 69 4.2.2.7. RKMT_176 metabolites production ............................................................ 71 4.2.2.8. Comparison of metabolites production between strains ............................. 72 4.2.2.9. Cluster analysis and barcode ....................................................................... 73 4.2.3. Biotransformations in bacterial isolates ............................................................ 75 4.2.3.1. Identification of the bacterial strain ............................................................ 75 4.2.3.2. Comparison of samples by LC-HRMS analysis ......................................... 76 4.2.3.3. Isolation and identification of compounds .................................................. 76 4.2.3.4. NMR analysis .............................................................................................. 78 5. Discussion ........................................................................................................................ 81 5.1. Mass spectrometry study of the starfish Luidia senegalensis….………..…………..81 5.2. Marine bacteria ........................................................................................................... 82 5.2.1. Cultivable bacterial communities of marine sediment and invertebrates .......... 82 5.2.2. Metabolomic study of the marine actinomycete Verrucosispora sp. .................. 85 5.2.3. Biotransformations in bacterial isolates ............................................................ 87 6. Conclusion ....................................................................................................................... 89 References ................................................................................................................................ 90 Supplementary Material ...................................................................................................... 101 23 1. Introduction 1.1. The by-catch fauna and marine invertebrates Current selective and rational fishing practices involve avoiding prohibited species and those with no commercial value. However, equipment required to catch high-value species such as fish and shrimp and the lack of robust markets for many unintentionally harvested marine organisms, commonly referred to as by-catch, makes the practical application of selective fishing practices challenging (HALL; ALVERSON; METUZALS, 2000). By-catch fauna is defined as any organism that is not the intended target of the harvest, including fishes, turtles, crustaceans, mollusks and other organisms. By-catch is a global problem, and the Food and Agriculture Organization (FAO) estimates that nearly 7 million tons of by-catch are discarded annually, an amount equivalent to 8% of the world’s marine fisheries. The by- catch associated with shrimp trawling in tropical waters is particularly egregious, accounting for 28% of all by-catch (EAYRS, 2007). Bottom trawling fishing practices not only leads to low harvest selectivity, but also the destruction of the neritic biological diversity, particularly in the demersal-benthic layer. In spite of high by-catch rates, the marketable portion of the catch is sufficiently profitable that current fishing practices remain profitable. Continued use of non-selective fishing practices reduce biodiversity as the majority of discarded by-catch perishes, thereby disrupting the ecological balance of fished areas (GRAÇA LOPES et al., 2002; SANTOS, 2007; SEVERINO-RODRIGUES; HEBLING; GRAÇA-LOPES, 2004). The broad variety of marine organisms that are caught together with fish and shrimp is completely ignored as a source of new molecules. Due to the low economic value, the fishermen despise them, because they are not marketable as food. However, several substances found in marine organisms can be widely applied in many areas to improve the quality of life of humanity, including the areas of food, power generation and medicine. Therefore, we investigate the chemical compounds present in the by-catch fauna of the shrimp fishery on the coast of State São Paulo, Brazil, in order to obtain molecules of potential economic interest, which can increase the value of this wasted material (CATTANI et al., 2011; CLUCAS, 1997). This approach is not the ideal since this predatory activity should be banned, but until now no other alternative for the shrimp fishery was found. This work does not intend to solve the by-catch problem, but rather use the material that is usually discarded. 24 To start our work on the chemical composition of the by-catch fauna of the shrimp fishery, we have investigated the polar extract of the nine-armed starfish Luidia senegalensis Lamark collected in the coast of the São Paulo State, Brazil. Luidia Forbes (1839, Luidiidae, Asteroidea: Paxillosida) are bottom sea stars, which live in sandy or muddy substrate. The genus includes 49 species that occur in tropical and subtropical shallow waters (XIAO et al., 2013). Luidia senegalensis [syn. Asterias senegalensis Lamarck (1816), Luidia marcgravii Steenstrup in Lutken (1859, synonym according to Perrier (1875)] occurs at depths of up to 40 meters alongside the coast of South America, including southern Brazil, as well as around the coasts of Florida, in the Caribbean Sea and the Gulf of Mexico (CLARCK; DOWNEY, 1992). Starfish (called also sea stars) are marine invertebrates widely recognized as source of natural products. They belong to the class Asteroidea, phylum Echinodermata. The chemical composition of a number of starfishes has been investigated using several chromatographic and spectrometric techniques. This class of Echinodermata is rich in free polyhydroxysteroids and two main groups of steroid glycosides: asterosaponins and the glycosides derived from the polyhydroxysteroids. The asterosaponins present a ∆9,11-3β,6α-steroidal core, with four rings, a sulphate group at C-3, one or two oxygenated carbons at the side chain, and four to six sugar moieties attached to C-6. Common saccharide residues are pentoses (xylose, arabinose), deoxyhexoses (quinovose, fucose), hexoses (glucose, galactose) and 6-deoxy- xylo-hex-4-ulose (DXHU). Therefore, the extracts produced from these marine organisms are often very complex mixtures of free and sulphated highly oxygenated compounds as well as their sodium salts. These sulphated steroid oligoglycosides usually have molecular weight higher than 1200 Da and may include isomeric compounds. These substances have a wide variety of pharmacological activities. Among them, they act as anti-viral, anti-bacterial, anti- inflammatory, anti-fungal, hemolytic, activate tubulin polymerization, inhibit tumour proliferation and possess immunomodulatory activities. They are also involved in physiological and chemical defense, interspecific chemical communication, digestion and reproduction (DATTA; TALAPATRA; SWARNAKAR, 2015; DONG et al., 2011). Recently, mass spectrometry coupled or not to liquid chromatography has been proved to be a powerful tool for the investigation of the saponins present in the polar extracts of sea stars, without the need of prior separation (DEMEYER et al., 2014; POPOV et al., 2014). In our case, the analysis of the polar hydroethanolic extract of L. senegalensis was accomplished using a combination of solid-phase extraction (SPE), direct flow injection- electrospray-ion trap tandem mass spectrometry (DFI-ESI-IT-MSn) and an ultra-high 25 performance liquid chromatography-electrospray-ion trap tandem mass spectrometry (UPLC- ESI-IT-MSn) method. Substances were tentatively identified using the typical fragmentation of the aglycone side chain, the characteristic losses of the sugar moieties and comparison with the fragmentation pattern described in the literature (DE MARINO et al., 2003; DEMEYER et al., 2014; DONG et al., 2011; MINALE et al., 1985; POPOV et al., 2014). 1.2. Marine bacteria and its biotechnological potential While the value of by-catch as a food-source is limited, many benthic organisms harvested as by-catch have value as sources of secondary metabolites with a wide range of biological activities. Many invertebrates also harbor a plethora of microorganisms, which also have the potential to produce novel bioactive compounds (BLUNT et al., 2015). In some cases, symbiotic microorganisms are the true producers of metabolites first isolated from marine invertebrates (MINCER et al., 2002). Microorganisms are widely recognized as one of the most important sources of bioactive natural products, many of which have found utility as treatments for a variety of diseases such as infectious diseases, cancer, hypercolesterolemia etc.(FISCHBACH; WALSH, 2009; HAYGOOD et al., 1999; HONG et al., 2009; SPELLBERG et al., 2008). Despite the awareness of the biomedical potential of microbial natural products, the first natural product isolated from a microorganism discovered by Brazilian researchers was reported only in 2000. Among microbial natural products discovered in Brazil, only a small percentage (9%) were discovered from bacteria (IÓCA; ALLARD; BERLINCK, 2014). Thus bacteria from Brazilian habitats represent an underexplored resource. Antibiotic-resistant pathogens are an increasing health threat that makes the need to find new antibiotics particularly urgent (KIM; KSHETRIMAYUM; GOODFELLOW, 2011). More than two-thirds of clinically used antibiotics are natural products or their semisynthetic derivatives. Despite this need for new bioactive compounds, natural product discovery has declined in the last few years, in part because of the rediscovery of known compounds and the difficulty in finding new antibiotics. Recent efforts to reinvigorate the antibiotic discovery via bioprospecting from underexplored ecological niches, unexplored bacterial taxa, and even the genomes of well-studied bacteria have yielded novel antimicrobial natural products, whereas new screening strategies have begun to circumvent the time consuming problem of rediscovery (FISCHBACH; WALSH, 2009). 26 Most natural product antibiotics have been discovered from bacteria within the order Actinomycetales (commonly referred to as actinomycetes), which are common soil inhabitants. Though more than 50% of the microbial antibiotics discovered so far originate from actinomycetes, two genera (Streptomyces and Micromonospora) account for most of these compounds (PROCÓPIO et al., 2012; WAGMAN; WEINSTEIN, 1980). Recent explorations of marine environments have established marine bacteria, including marine actinomycetes, as a promising source of bioactive natural products (EL AMRAOUI et al., 2014). This fact is exemplified by the discoveries of the potent anticancer agent salinosporamide A and the novel antibiotic abyssomycin C from the marine actinomycetes Salinospora tropica and Verrucosispora sp., respectively (BISTER et al., 2004; NIEWERTH et al., 2014). Two marine species commonly harvested as by-catch in bottom trawling fishing practices are the starfish Luidia senegalensis (Lamark, 1816) (CLARCK; DOWNEY, 1992) and the gastropod Ollivancilaria urceus (Röding, 1798) (BOLTEN, 1906). To add potential value to these by-catch organisms and to expand the breadth of Brazilian research in the area of bacterial natural product discovery we set out to study the culturable bacteria associated with L. senegalensis and O. urceus for their ability to produce bioactive natural products. A marine sediment sample collected in the same area as the invertebrates was also examined as sediments are an established source of natural product-producing marine bacteria (DALISAY et al., 2013; GONTANG; FENICAL; JENSEN, 2007; SPONGA et al., 1999). Due to their proven track record as a rich source of novel natural products we focused our isolation methods on those selective for actinomycetes. Isolated bacteria were identified by 16S rRNA gene sequencing and fermentation extracts from selected isolates were screened for antimicrobial activity. The chemical composition of bioactive extracts was characterized by liquid chromatography-high-resolution mass spectrometry to identify compounds potentially responsible for the observed bioactivity. 1.3. Metabolomics and the chemical study of marine bacteria The oceans covers 70% of earth’s surface and harbors most of the biodiversity of the planet (FENICAL; JENSEN, 2006), which can be related to a great molecular diversity of natural products found in animals, plants and microorganisms (KÖNIG et al., 2006). Such 27 environment presents a role set of factors that affect its inhabitants like pressure, salinity, temperature and nutrient availability (BOSE et al., 2015), which may affect their metabolism. Among all the living beings from the seas, microorganisms stand out for their capacity to thrive in several marine environments, from the water surface until the lower and abyssal depths, from coastal to offshore regions and from the general oceanic to the specialized niches (DAS; LYLA; KHAN, 2006). This fact shows the high adaptability of these organisms, due to their genetic plasticity and rapid replication, making microbes to be the most numerous, diverse and adaptable organisms on earth (SPELLBERG et al., 2008). This organisms have drawn the attention of scientists for many years due to their importance in many life processes, detrimentally or in the production of useful compounds such as vitamins, antibiotics and other pharmaceuticals (IÓCA; ALLARD; BERLINCK, 2014). In fact, almost 70% of small molecules that are utilized as medicines are derived or inspired in natural products produce by bacteria, more specifically filamentous actinobacteria (SEIPKE, 2015). When studying microorganisms, several parameters must be accessed to fully understand the production of secondary metabolites by one strain. For example, media composition, pH, temperature, oxygen availability and light intensity may affect microbes metabolism, therefore affecting compound production (RATEB et al., 2011). This parameters can be evaluated using the OSMAC (One Strain – Many Compounds) approach, where the alteration of culture conditions may turn on silent or cryptic biosynthetic genes and generate new metabolites where it is been reported from one single strain it could be isolated up to 20 different metabolites in yields up to 2.6 g L-1 (BODE et al., 2002; CHRISTIAN et al., 2005; RATEB et al., 2011). One of the major difficulties in natural products research is the rediscovery of known compounds. This problem can be avoided by the use of metabolomics, with hyphenated techniques to the fast and reliable evaluation of large sets of samples to prioritize the most promising sources of unknown compounds (HARVEY; EDRADA-EBEL; QUINN, 2015). The principal platforms utilized nowadays for metabolomics studies are LC-NMR and LC- MS assemblies, the second one being used in a larger proportion due to its lower cost and faster optimization and data processing (IBEKWE; AMEH, 2015). LC-MS based metabolomics is also a powerful tool in the study of known compounds production in different conditions (HARVEY; EDRADA-EBEL; QUINN, 2015). For example, seasonality studies in plant natural products (KWAK; HEGEMAN; PARK, 2014), chemical composition of marine invertebrates from different site collections and compound production of microorganisms (e.g. fungi, bacteria) submitted to different cultivation conditions (BOSE et 28 al., 2015). In this way, it is possible to find the best conditions for the production of bioactive compounds for better yields and time optimization. An interesting and rare genus of marine actinomycete, Verrucosispora, contains species that are a good example of promising producers of bioactive metabolites (FIEDLER et al., 2005). The genus comprises only eight species isolated from mangroves, summarized in Table 1. Table 1. Species belonging to Verrucosispora genus, location and source of first report Species Location Source Ref V. lutea Guangdong Province, China Mangrove sediment (LIAO et al., 2009) V. qiuiae Hainan Province, China Swamp sediment (XI et al., 2012) V. wenchangensis Wenchang, China Mangrove sediment (LIN; LI, 2012) V. gifthornensis Gifhorn, Germany Peat sample (RHEIMS et al., 1998) V. sediminis South China Sea Deep-sea sediment sample (DAI et al., 2010) V. maris East Sea of Japan Deep-sea sediment sample (GOODFELLOW et al., 2012) V. fiedleri Norway Fjord sediment (GOODFELLOW et al., 2013) V. andamanensis Phuket Province of Thailand Sponge Xestospongia sp. (SUPONG et al., 2013) Such organisms are great producer of bioactive compounds like the antibiotic and anticancer agents proximicins A, B and C (FIEDLER et al., 2008), produced by isolates of V. maris and V. fiedleri (GOODFELLOW et al., 2013; ROH et al., 2011), the diterpenes inhibitors of androgen receptors gifhornenolones A and B, produced by V. gifhornensis isolates (SHIRAI et al., 2010) and the potent antibiotic abyssomicins and its derivatives (BISTER et al., 2004; KELLER et al., 2007; WANG et al., 2013), from V. maris isolates and presenting a completely new scaffold, reinforcing the promising source of novel bioactive compounds from the marine environment. Also the cytotoxic thiodepsipeptide thiocoraline and its analogs were found to be produced by other Verrucosispora sp. isolates (WYCHE et al., 2011). Therefore, a study was carried out with three strains of Verrucosispora sp., using LC- HRMS based metabolomics for the detection and identification of secondary metabolite production. The three isolates were fermented in nine different media each and the resulting broth extracted with ethyl acetate and dried. For the evaluation of media composition effect in 29 compound production the extracts were analyzed by LC-HRMS followed by data processing and statistical analysis including cluster and principal component analysis (PCA). This methodology allows the rapid identification of compounds such as putatively new metabolites as well the fermentation conditions leading to good production yield. 1.4. Proteobacteria and biotransformations Another important feature of the marine bacteria is the capability of biotransformation, providing new derivatives of known compounds with potential different activity, without the need of time-consuming low-yield synthesis steps (LI et al., 2006). Biotransformations by microorganisms are an important tool in industrial processes (BOAVENTURA; LOPES; TAKAHASHI, 2004; RODRÍGUEZ‐GARCÍA et al., 2016). Among its advantages are the less drastic conditions applied compared to synthesis like neutral pH, ambient temperatures and atmospheric pressure (HEGAZY et al., 2015) and the factor that are usually carried out in aqueous systems, which avoids the use of harmful solvents common in synthesis (BOAVENTURA; LOPES; TAKAHASHI, 2004). Moreover biotransformations are, in general, regio- and stereoselective (BAYDOUN et al., 2014), a major advantage compared to common synthesis. Several microorganisms can be used in processes for biotransformations, such as fungi (GAUTHIER et al., 2016; HEIDARY; HABIBI, 2016; KOLLEROV et al., 2015), microalgae (GHASEMI; RASOUL-AMINI; FOTOOH-ABADI, 2011), yeasts (GORETTI et al., 2009) and bacteria (terrestrial and marine) (COLQUHOUN et al., 1998; HYLEMON; HARDER, 1998; LI et al., 2006). Marine bacteria are a singular group which shows unique characteristics like the production of salt-tolerant enzymes, which may be very useful in industrial processes (DEBNATH; PAUL; BISEN, 2007). The main group of marine bacteria explored for its genetic machinery is the actinomycetes (COLQUHOUN et al., 1998; ISHIHARA et al., 2011), but Proteobacteria also contains individuals with potential applications in biotransformations (LI et al., 2006). In fact, reports say that marine Proteobacteria may have a higher complement of enzymes than Actinobacteria, differently from their terrestrial analogs where the opposite occurs (MUHLING; JOINT; WILLETTS, 2013). Therefore a study was carried out with a Proteobacteria marine isolate, Erythrobacter vulgaris, isolated from a marine sediment sample from the southeast coast of Brazil, using 30 LC-HRMS, cultivation, chromatographic purification and NMR spectroscopy to identify compounds produced or possible biotransformations performed by the isolated strain. 31 2. General and Specific Goals The main goal of the study was to evaluate the chemical composition of marine invertebrate species from the by-catch fauna of shrimp fisheries in the São Paulo State. Also, investigate the marine bacteria associated to the invertebrates and sediment for its biotechnological potential. In brief, the specific goals of the study were: • Investigate the chemical composition of the starfish Luidia senegalensis in order to identify its secondary metabolites • Isolate and characterize the cultivable marine bacteria associated to the invertebrates and to the sediment collected • Assess the capability of the isolated bacteria concerning its biotechnological potential for compound production and/or biotransformations 32 3. Materials and Methods For this study, three beaches located in the north, central and south of the São Paulo State traditionally used for the commercial shrimp fishing were selected: 1) North: Ubatuba, Itaguá Beach (23°26'12"S, 23°27'78"S and 45°00'76"W at 45°02'94"W), 2) Central: in Guaruja, Perequê Beach (23°46'45"S at 23°58'07"S and 46°08'42"W at 46°09'33") and 3) South: Itanhaém, Praia dos Pescadores Beach (24°11'39"S at 24°12'48"S and 46°46'44"W at 46°47'54"W). In each locality, two trawls were performed, one in January (hot season, average temperature 21oC) and one in July (cold season, average temperature 19oC), in 2013. Local vessels were used, equipped with a semi-ballon otter trawl net, 2.0 cm in the collecting sac and bagger of 14 mm between adjacent nodes, at a mean speed of two knots and depths ranging between 10 - 20 m in 30 min each trawl. The salinity remained at approximately 33.0. The collected animals were previously separated by large taxonomic groups, placed in plastic bags and stored in Styrofoam box with ice for transporting to the laboratory. The lab activities consisted of thawing the material, sorting, identifying at the lowest possible taxonomic level according to the literature for each major group identified, measurement and weighing of specimens. Materials from the different sampling sites were analyzed separately. 3.1. Mass spectrometry study of the starfish Luidia senegalensis 3.1.1. Chemicals and Materials Methanol Chromasolv LC–MS grade, chloroform, 1-propanol, formic acid, ninhidrin, anysaldehyde and sulfuric acid were acquired from Sigma-Aldrich (São Paulo, Brazil). Ultrapure water was produced using a Milli-Q system (Millipore, Bedford, MA, USA). 3.1.2. Animal material, extraction and fractionation Individuals of Luidia senegalensis were collected in the city of Ubatuba – SP, The specimens were kept in ice for transportation and frozen after arriving in the laboratory until extraction. The starfish was identified by Prof. Dr. Tania Marcia Costa, from UNESP - 33 Coastal campus of São Vicente. In addition, the specimens were photographed to afford a visual voucher. The animals were thawed to room temperature, separated by species, crushed with mortar and pestles. One gram of each animal was extracted by maceration with 10 mL of ethanol 70% (3 days). The extract was filtered and the solvent was removed under vacuum at 40 °C, using a rotary evaporator. The SPE cartridge (500 mg, Macherey-Nagel, Chromabond C18 ec, Düren, Germany) was first preconditioned by the consecutive passing of 5 mL of methanol and then 5 mL of pure water by gravity. The extract obtained was solubilized in water at concentration of 1.0 mg mL-1, filtered on filter paper to remove macro particles and further filtered through a 0.45 µm membrane of a PTFE filter. The sample was loaded to the cartridge and first eluted with 5 mL of water (to eliminate salts and free amino acids). The cartridge was eluted again with 5 mL of water/methanol (90:10, v/v, named hydromethanolic fraction - HF) and finally with 5 mL of pure methanol. All three fractions were analyzed using two thin layer chromatography plates (TLC, silicagel, 20 x 20 cm, 250 µm layer, UV fluorescence 254 nm, Whatman Ltd, Maidstone, England; Eluent: chloroform:methanol:n-propanol:water 100:11:11:27 v/v) and separately revealed with ninhidrin (to detect amino acids) and anisaldehyde/sulfuric acid mixture (to detect secondary metabolites). Fraction collected in water contained only amino acids and other impurities and HF concentrated the compounds of interest. HF was transferred into clean tubes and dried under nitrogen gas at room temperature. The sample was redissolved in pure methanol/water 50:50, v/v to a concentration of 5 ppm and analyzed by mass spectrometry. 3.1.3. ESI-MS n analysis Direct flow infusion of the samples was performed on a Thermo Scientific LTQ XL linear ion trap analyzer equipped with an electrospray ionization (ESI) source, both in positive and negative modes (Thermo, San Jose, CA, USA). It was used a fused-silica capillary tube at 280 oC, spray voltage of 5.00 kV, capillary voltage of -35 V, tube lens of -100 V and a 5.0 µL min-1 flow. Full scan analysis was recorded in m/z range from 100-2000. Multiple-stage fragmentations (ESI-MSn) were performed using the collision-induced dissociation (CID) method against helium for ion activation. The first event was a full-scan mass spectrum to acquire data on ions in that m/z range. The second scan event was an MS/MS experiment performed by using a data-dependent scan on the desodiated molecules from the compounds 34 of interest at a collision energy of 30% and an activation time of 30 ms. The product ions were then submitted to further fragmentation in the same conditions, until no more fragments were observed. 3.1.4. UPLC-ESI-IT-MS analysis UPLC-ESI-IT-MS analysis were carried out in a Thermo Scientific® ultra- performance liquid chromatography equipment, consisting of an Accela AS autosampler, a quaternary Accela pump 600 coupled with the LTQ XL mass spectrometer described above, operating under the same conditions. Chromatographic separations were performed on a non- polar column (Kinetex® core-shell, C18, 1.7 µm, 100 x 2.1 mm, Phenomenex, USA) at room temperature. The mobile phases consisted of eluent A (0.1% formic acid in water, v/v) and eluent B (0.1% formic acid in methanol, v/v). These eluents were delivered at a flow rate of 0.2 mL min-1 with a linear gradient program as follows: 40–100% B from 0 to 5.0 min. After maintaining 100% B for 5 min, the column was returned to its initial condition. Aliquots of 10 µL of the samples were injected into the UPLC-ESI-IT-MS system for analyses using the autosampler. In the UPLC-ESI-IT-MSn experiments for each parent ion of m/z 1405, m/z 1389, m/z 1239 and m/z 1227 we used the four product ions obtained after MS2, MS3 and MS4 experiments and CID of 30 eV against each ion, using the same chromatographic conditions. 3.2. Marine bacteria 3.2.1. Cultivable bacterial communities of marine sediment and invertebrates 3.2.1.1. Sample collection The marine bacteria studied was isolated from the gastropod Olivancillaria urceus and the starfish Luidia senegalensis collected of the coast of Ubatuba – state of São Paulo, Brazil, as previously described. The sediment sample was collected at the end of the trawl using a van Veen grab sampler deployed from the fishing vessel. The sediment sample was kept wet with sea water and stored in on ice until arrival in the laboratory. In the laboratory, it was kept refrigerated at 4 ºC until processing. 35 3.2.1.2. Methods for the isolation of bacteria For the isolation of bacteria, four different pretreatments specific to each type of sample were performed. When seawater was needed, seawater collected from the northern coast of Prince Edward Island, Canada, was filtered over a 0.45 µm cellulose nitrate membrane and autoclaved. Serial dilutions were prepared using sterile sea water. Isolation plates were incubated at 28 ºC for a period of 10 days. (1) Dry-stamp method (dry sediment) (MINCER et al., 2002). A portion (5 g) of wet sediment was dried aseptically in a sterile petri dish (24 h, 37 ºC) and ground lightly with a sterile mortar and pestle. An autoclaved foam plug (diameter 10 mm) was pressed into the ground sediment and excess sediment was dislodged from the plug by gently tapping the edge of the plug. Agar plates were inoculated by stamping the foam plug on the surface of the plate in a circular fashion resulting in a serial dilution effect. Three plates each of media 1-5 were inoculated in this fashion. (2) Dispersial and differential centrifugation technique (DDC - wet sediment) (HOPKINS; MACNAUGHTON; O’DONNELL, 1991). 5.0 g of wet sediment were ground with a sterile mortar and pestle in 10.0 mL of 0.1% (w/v) sodium cholate solution. The ground sediment was aseptically transferred to a 50 mL centrifuge tube and an additional 10 mL of 0.1% (w/v) sodium cholate solution was added along with 30 sterile glass beads (4 mm diameter). The tube was shaken on its side at 60 rpm at 5 °C for 2 h. The mixture was centrifuged at 500g for 2 min and the supernatant saved (DDC1). The precipitate was suspended in 10 mL of 50 mM Tris-HCl buffer (pH 8.0) and stirred for 1 hour at 5 oC. The mixture was then centrifuged at 500g for 1 min. The supernatant was combined with DDC1. The pellet was suspended in 20 mL sodium cholate solution and sonicated in a Misonix Sonicator 3000 (New York, USA) at low power for 1 min. Following sonication 10 mL of sodium cholate solution was added and the tube was shaken on its side at 60 rpm for 1 h at 5 °C. The mixture was centrifuged at 500g for 1 min and the supernatant (DDC2) saved. The pellet was suspended in 10 mL of 50 mM Tris-HCl buffer (pH 8.0) and shaken on its side at 60 rpm for 1 h at 5 oC. The mixture was centrifuged at 500g for 1 min and the supernatant combined with DDC2. Cells present in DDC1 and DDC 2 were collected by centrifugation (12,000g, 4 °C), and suspended in 10 mL of 50 mM Tris-HCl buffer. For media 1-5 0.1 mL of undiluted and a 10-1 dilution of DDC1 and DDC2 were plated on five plates of each medium. For Marine Agar (MA), DDC1 and DDC2 were serially diluted (10-1 to 10-7) and 0.1 mL of each dilution was spread on three plates per dilution. 36 (3) Heat shock method (invertebrates) (MINCER et al., 2002). Invertebrates were thawed at room temperature and in the case of O. urceus shells were aseptically removed once thawed. From the starfish, two arms and the center disc of one specimen were homogenized. From O. urceus, two entire specimens were homogenized. Both invertebrates were mixed with 9 mL of sterile seawater and blended in a stainless steel autoclaved waring blender. The mixture was heated for 60 min at 55 °C to reduce the viability of asporogenous bacteria and then vortexed for 1 minute. The homogenates were serially diluted (10-1 to 10-8) in sterile seawater and 0.1 mL of each dilution was plated on agar plates. For media 1-5, the 10-1 to 10-3 dilutions were plated on each medium in triplicate. For MA, 0.1 mL of each dilution (10-1 to 10-8) was plated in triplicate. (4) Phenol method (invertebrates) (HAYAKAWA; YOSHIDA; IIMURA, 2004). Phenol (1.5% v/v) was added to 10 mL of each invertebrate homogenate and shaken at 200 rpm for 30 minutes. Phenol-treated homogenates were diluted and plated as described for method 3. Bacteria were also isolated from invertebrate homogenates without pretreatment. For these samples a portion (0.1 mL) of serially diluted homogenates (10-1 to 10-8) were plated on triplicate plates of MA. 3.2.1.3. Culture media for bacterial isolation Five media were used, which had previously been developed for the isolation of Actinobacteria. The compositions of the media are listed below. Culture Medium 1 (HAYAKAWA; NONOMURA, 1987) (per L): humic acid, 1.0 g (solubilized in 10.0 mL NaOH 0.2 N); Na2HPO4, 0.5 g; KCl, 1.71 g; MgSO4•7H2O, 0.05 g; FeSO4•7H2O, 0.01 g; CaCO3, 0.02 g; vitamins (thiamine-HCl, riboflavin, niacin, pyridoxine- HCl, inositol, Ca-pantothenate and p-aminobenzoic acid, 0.5 mg each, and 0.25 mg of biotin); agar, 18.0 g; seawater, to 1.0 L; pH 8.0; Vitamins were filter sterilized (0.22 µm) and added to the medium after autoclaving. Culture Medium 2 (ROWBOTHAM; CROSS, 1977) (per L): KH2PO4, 0.466 g; Na2HPO4, 0.732 g; KNO3, 0.10 g; MgSO4•7H2O, 0.10 g; CaCO3, 0.02 g; sodium propionate, 0.20 g; FeSO4•7H2O, 200 µg; ZnSO4•7H2O, 180 µg; MnSO4•H2O, 20 µg; thiamine•HCl 4 mg; agar, 18.0 g; seawater, to 1.0 L; pH 8.0. Thiamine was filter sterilized (0.22 µm) and added to the medium after autoclaving. Culture Medium 3 (ATHALYE; LACEY; GOODFELLOW, 1981) (per L): yeast extract, 10.0 g; glucose, 10.0 g; agar, 15.0 g; seawater, to 1.0 L; pH 8.0. Culture Medium 4 (VICENTE et al., 2013): 37 mannitol, 0.5 g; peptone, 0.1 g; agar, 18.0 g; seawater, to 1.0 L; novobiocin (20 mg) was filter sterilized and added post sterilization. Culture Medium 5 (VICENTE et al., 2013) (per L): colloidal chitin, 0.5 g; agar, 18.0 g; seawater to 1.0 L. Marine Agar (Difco TM ) (MA) was prepared with deionized water according to the manufacturer’s recommendations. Cycloheximide (50 mg L-1) was added to media 1, 2, 4, and 5 to suppress fungal growth (WILLIAMS; DAVIES, 1965). Rifampicin (5 mg L-1) and streptomycin (15 mg L-1) were added to medium 3 to suppress the growth of fast-growing Streptomyces and to favor the isolation of less frequently isolated Actinobacteria such as Actinoplanes (DWORKIN et al., 2006). Novobiocin (20 mg L-1) was added to media 4 and 5 to suppress the growth of non- actinomycetes (QIU; RUAN; HUANG, 2008). Emerging colonies were purified by serial subculturing on the same medium from which they were first observed. Following initial purification isolates were transferred to MA culture medium and those exhibiting similar morphological characteristics from the same source were grouped and one of each group was selected for identification. 3.2.1.4. Bacteria identification Bacteria were identified by sequencing of the small subunit (16S) ribosomal RNA gene. To generate template DNA for PCR amplification one colony of each axenic strain was suspended in 50 µL PCR grade DMSO (Sigma). PCR amplification of the 16S rRNA gene was conducted in 50 µL volumes and consisted of the following: EconoTaq® PLUS GREEN 2X Master Mix (25 µL) (Lucigen, Middleton, WI, USA), 0.5 µM of the primers pA (5’- AGAGTTTGATCMTGGCTCAG) and pH (5’-AAGGAGGTGWTCCARCC) and genomic DNA (2.5 µL of template in DMSO) (EDWARDS et al., 1989). Thermal cycling parameters consisted of initial denaturation at 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1.5 min followed by a final extension at 72 °C for 5 min. A negative control, which lacked template DNA (DMSO only), was included in each set of PCR reactions. Amplification was evaluated by agarose gel electrophoresis. Partial sequencing of 16S rDNA amplicons was performed by Eurofins MWG Operon (Huntsville, AL, USA) using the 16S936R primer (5′-GGGGTTATGCCTGAGCAGTTTG) (DUNCAN et al., 2014). 38 3.2.1.5. Phylogenetic analysis of cultured bacteria Sequences were analyzed, edited and grouped into operational taxonomic units (OTUs) using the Contig Express application within the Vector NTI version 10.3 software package (Invitrogen, Carlsbad, CA, USA). The level of 16S rDNA sequence identity which corresponds to genomic species demarcation based on a genome average nucleotide identity of 95-96% was recently determined to be 98.65% (KIM et al., 2014). This determination was based on nearly full-length 16S rDNA sequences. As we obtained partial sequences approximately 600-800 bp in length, a more conservative species level 16S rDNA identity cut-off of 99% identity was used to define OTUs. To identify the closest relatives, sequences were compared to those in the NCBI database (http://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST) (ALTSCHUL et al., 1990). Sequences were aligned using BioEdit version 7.2.5 with the ClustalW tool and phylogenetic analysis of partial 16S rDNA sequences was conducted using the neighbor-joining algorithm (SAITOU; NEI, 1987) based on distances estimated by Kimura's two-parameter model using Molecular Evolutionary Genetics Analysis - MEGA Version 6.0 (TAMURA et al., 2013). Neighbour-joining (NJ) trees were prepared using default settings with complete deletion (FELSENSTEIN, 1985). The robustness of the resulting phylogeny was evaluated by bootstrap analysis of NJ data based on 1000 re-samplings (FELSENSTEIN, 1985). 3.2.1.6. Bacteria cultivation and extraction Marine Broth (DifcoTM) medium was prepared with Milli-Q water according to the manufacturer’s recommendations and dispensed (7 mL) into culture tubes (150 × 25 mm) containing 3 glass beads (4 mm dia) and sterilized by autoclaving (121 °C for 30 min). Seed cultures were prepared by inoculating an axenic colony into a fresh tube and culturing for three days at room temperature (22-25 °C) and 200 rpm. After this period, 1.0 mL of the broth was transferred to a new tube containing the same medium, which was fermented for one more day under the same conditions. A portion (210 µL) of the second stage seed culture was used to inoculate duplicated culture tubes containing 7 mL of Marine Broth. Tubes were incubated at room temperature and 200 rpm for 7 days. Fermentations were extracted twice with 10 mL ethyl acetate by rapid agitation (200 rpm) at room temperature for one hour. Ethyl acetate extracts were washed with Milli-Q water to remove salts, and then dried under air. 39 3.2.1.7. Antimicrobial assays Antimicrobial assays were performed by Martin Lanteigne from the University of Prince Edward Island – UPEI. All microbroth dilution antimicrobial screening was carried out in 96-well plates in accordance with Clinical Laboratory Standards Institute testing standards (NCCLS, 2003) using the following pathogens: methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), S. warneri ATCC 17917, vancomycin-resistant Enterococcus faecium EF379 (VRE), Pseudomonas aeruginosa ATCC 14210, Proteus vulgaris ATCC 12454, and Candida albicans ATCC 14035. Extracts were dissolved in sterile 20% DMSO (aq) and assayed at 100 µg mL-1 with a final DMSO concentration of 2% DMSO (v/v; aq). Each plate contained eight uninoculated positive controls (media + 2% DMSO (aq)), eight untreated negative controls (Media + 2% DMSO(aq) + organism), and one column containing a concentration range of a control antibiotic (vancomycin for MRSA, and S. warneri, rifampicin for VRE, gentamicin for P. aeruginosa, ciprofloxacin for P. vulgaris, or nystatin for C. albicans). The optical density (OD) of the plate was recorded using a Thermo Scientific Varioskan Flash plate reader at 600 nm at time zero and then again after incubation of the plates for 22 h at 37 °C. After subtracting the time zero OD600 from the final reading, the percentages of microorganism survival relative to vehicle control wells were calculated. 3.2.1.8. UPLC-HRMS analysis Extracts that showed antimicrobial activity were analyzed by liquid chromatography– mass spectrometry analysis, carried out with an ESI-HRMS Thermo Scientific® EXACTIVE operating on positive mode with a resolution of 30,000, monitoring a mass range from 200 to 2000 atomic mass units (amu), using a Core-Shell 100 Å C18 column (Phenomenex® Kinetex, 1.7 µm, 50 × 2.1 mm). A linear solvent gradient from 95% H2O/0.1% formic acid (solvent A) and 5% CH3CN/0.1% formic acid (solvent B) to 100% solvent B over 4.8 min followed by a hold for 3.2 min with a flow rate of 500 µL/min and 10 µL injection volume was used. The mass spectrometer was hyphenated to an ELSD and UV detector (200 − 600 nm). All analyses were processed using Thermo Xcalibur 2.2 SP1.48 software. To putatively identify ions observed in LC-HRMS analyses, observed molecular weights of pseudomolecular ions were used to search the Antibase 2014 (Wiley-VCH) database. To avoid interference from media components, media blanks were analyzed using the same method and the ions observed subtracted from the sample files. 40 3.2.2. Metabolomic study of the marine actinomycete Verrucosispora sp. 3.2.2.1. Bacteria isolation and identification Metabolomics study assessed the metabolite production of 3 strains of Verrucosispora sp. isolated from samples collected in the coast of Ubatuba – SP. Strains RKMT_073 and RKMT_111 were isolated from a sediment sample using dry-stamp method (MINCER et al., 2002). Strain RKMT_176 was isolated from a sea star, Luidia senegalensis, using Phenol method (HAYAKAWA; YOSHIDA; IIMURA, 2004). The isolation of RKMT_073 was achieved using Cultivation Medium 2 (ROWBOTHAM; CROSS, 1977) and RKMT_111 and RKMT_176 were isolated using Cultivation Medium 5 (VICENTE et al., 2013), both previously described. All plates were incubated at 30 ºC. When seawater was needed, seawater collected in the northern coast of Prince Edward Island, Canada, was filtered over a 0.45 µm cellulose nitrate membrane and autoclaved. Identification was made by full sequence of the 16S rDNA. Partial sequencing of 16S rDNA amplicons was performed by Eurofins MWG Operon (Huntsville, AL, USA) using the primers 16S936R (5′-GGGGTTATGCCTGAGCAGTTTG), 16S1114F (5′-GCA ACGAGCGCAACCC), 16S530R (5′-GTATTACCGCGGCTGCTGG), 16S27F (5’- AGAGTTTGATCM TGGCTCAG) and 16S1525R (5’-AAGGAGGTGWTCCARCC). Sequences were manually corrected using Chromas Lite 2.1.1 Technelysium Pty Ltd, assembled with online CAP3 Sequence Assembly Program (http://doua.prabi.fr/software/cap3) and the final sequences (~1500bp) compared within the NCBI database sequences (http://www.ncbi.nlm.nih.gov/). The comparison was made by pairwise distance estimation analysis, using Kimura 2 – parameter model in MEGA6 software (TAMURA et al., 2013). All sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) library (Pending Accession Number). 3.2.2.2. Bacteria fermentation Three Verrucosispora sp. strains, RKMT_073, RKMT_111 and RKMT_176, were cultivated in nine different types of liquid media each, in duplicate at 30ºC, to evaluate their capacity in the production of secondary metabolites. Thus, the following composition of media was utilized. (1) BFM-1m (LIU; SHEN, 2000) – Dextrin, 20.0 g; Soluble starch, 20.0 g; Beef extract, 10.0 g; Peptone, 5.0 g; (NH4)2SO4, 2.0 g; CaCO3, 2.0 g; Synthetic sea salt 41 (Instant Ocean), 18.0 g and deionized H2O, 1.0 q.s./L. (2) BFM-2m (NAGAOKA et al., 1986) – Soluble starch, 5.0 g; Pharmamedia, 5.0 g; Synthetic sea salt (Instant Ocean), 18.0 g and deionized H2O, 1.0 q.s./L. (3) BFM-3m (GRAZIANI et al., 2005) – MgSO4·7H2O, 0.5 g; KCl, 0.5 g; K2HPO4, 3.0 g; NaCl, 5.0 g, Agar, 0.4g; Glycerol, 12.0 g; Soy peptone, 5.0 g; Synthetic sea salt (Instant Ocean), 18.0 g; Deionized H2O, 1.0 q.s./L; (4) BFM-4m – Nutrisoy, 12.0 g; NH4Cl, 1.0 g; Dextrose, 12.0 g; Agar, 0.4 g; CaCO3, 1.0 g; NZ-amine A, 3.0 g; Synthetic sea salt (Instant Ocean), 18.0 g and deionized H2O, 1.0 q.s./L. (5) BFM-5m – Pancreatic Digest of Casein, 17.0 g; Enzymatic Digest of Soybean Meal, 3.0 g; NaCl, 5.0 g; K2HPO4, 2.5 g; Dextrose, 2.5 g; Synthetic sea salt (Instant Ocean), 18.0 g and deionized H2O, 1.0 q.s./L. (6) BFM-11m (JENSEN et al., 2007) – Starch (potato), 10.0 g; Yeast Extract, 4.0 g; Peptone, 2.0 g; KBr stock (20 g L-1), 5.0 mL; FeSO4·7H2O (8 g L-1, pH 7), 5.0 mL; Synthetic sea salt (Instant Ocean), 18.0 g and deionized H2O, 1.0 q.s./L. (7) ISP3m – Oat meal, 20.0 g (boiled for 20 min); Trace salts solution (FeSO4·7H2O, 0.1 g, MnCl2·4H2O, 0.1 g, ZnSO4·7H2O in 100 mL of deionized H2O), 1.0 mL; Synthetic sea salt (Instant Ocean), 18.0 g and deionized H2O, 1.0 q.s./L. (8) ASW-Am (WYCHE et al., 2011) – Soluble starch, 20.0 g; Glucose, 10.0 g; Peptone, 10.0 g; Yeast extract, 5.0 g; CaCO3, 5.0 g; Synthetic sea salt (Instant Ocean), 18.0 g and deionized H2O, 1.0 q.s./L. (9) Marine Broth (DifcoTM and BBLTM) – Peptone, 5.0 g; Yeast extract, 1.0 g; Iron citrate (III), 0.1 g; NaCl, 19.45 g; MgCl, 8.8 g; Na2SO4, 3.24 g; CaCl2, 1.8 g; KCl, 0.55 g; NaHCO3, 0.16 g; KBr, 0.08 g; SrCl2, 34.0 mg; H3BO3, 22.0 mg; Na2SiO3, 4.0 mg; NaF, 2.4 mg; NH4NO3, 1.6 mg; Na2HPO4, 8.0 mg and deionized H2O, 1.0 q.s./L. The seed medium was prepared first by inoculation of a CFU (colony forming unit) of each isolate in a tube containing 7.0 mL of ISP3m liquid medium and fermented for 3 days at 30ºC. Then, 1.0 mL of the fermentation broth was inoculated in a fresh tube containing 7.0 mL of the same liquid medium and fermented for one extra day. Finally, all media for the study (7.0 mL) were inoculated with 210 µL (3% of broth volume) of the fermentation broth and cultivated at 30ºC during 7 days. 3.2.2.3. Extraction and LC-HRMS analysis All fermentation broths were extracted with two portions of 10.0 mL of ethyl acetate, for 1 hour each time, at 200 rpm. After extraction, all samples were washed with two portions of 5.0 mL of deionized water, the ethyl acetate fractions were dried under air flow and the resulting material diluted to 500 µg mL-1 in methanol. Liquid chromatography–mass 42 spectrometry analyses were carried out with an ESI-HRMS Thermo Scientific® EXACTIVE operating on positive mode as previously described. 3.2.2.4. Preprocessing and statistical analysis LC−HRMS profiles were analyzed using PCA (principal component analysis) as previously described (FORNER et al., 2013). Briefly, after LC-HRMS analysis, the files were converted from .RAW (proprietary format) to .CDF (non-proprietary format) extension using XCalibur software. All the files were then processed using MZmine 2 (PLUSKAL et al., 2010), where the converted files were submitted to the steps of mass detection, chromatogram building, deisotoping, alignment and exportation. Mass detection step generates a list of exact masses for each scan in the analysis. For this step, it was chosen a noise level value of 1.0x104 counts s-1 by comparison with methanol and media blanks. Peaks presenting a lower intensity were therefore not detected and not included in the mass list for further analysis. The next step, chromatogram building, builds a chromatogram for each mass that can be detected in all scans from the mass list generated in the previous step. Then, deisotoping step was carried out for the selection of the representative ions in isotopic patterns. The last step in MZmine 2 software was the alignment mode, where m/z and retention time (rt) data were combined and all samples aligned in a single file. The alignment mode allows the conversion of a three dimensional dataset to a two dimensional dataset, where buckets are created with the peaks intensity. For the peaks that are not present in the sample, a value of zero was assigned. Finally, the aligned peaks were exported as a .csv (comma separated value) file. Standardization and artifact suppression were carried out in Microsoft Excel 2010. First, a presence-absence standardization was achieved transforming the dataset in a binary pattern, where intensities greater than zero were given a value of one and intensities equal to zero remained zero. For the easy visualization and pattern recognition, values of “1” within the dataset are shown as black, while values of “0” are white. Ions detected in MeOH and media blanks were removed from samples for media and artifact suppression. Ions that were not consistent in both replicates were excluded. Statistical analysis was carried out using The Unscrambler (Camo software). Principal component analysis was performed for comparison and for identification of putatively new compounds. Cluster analysis was performed using Ward’s Method and Squared Euclidean distances, as previously described (FORNER et al., 2013; WARD, 2011). 43 The number of compounds was estimated considering the detected adducts with approximate retention time. When no adduct was detected, the ion was considered as one compound. All adducts corresponding to each compound are shown in Table S1 – Supplementary Material. 3.2.2.5. Identification of compounds After detection and recognition of adducts the ions were compared in Antibase 2012 Wiley® (LAATSCH, 2012) through search of the [M+H]+ and [M+Na]+. Identification of known metabolites was further confirmed using the Scifinder database (http://www.scifinder.cas.org) and searching by molecular formula (Table S1 – Supplementary Material – Page 107). 3.2.3. Biotransformations in bacterial isolates 3.2.3.1. Bacteria isolation and identification Strain RKMT_070 was isolated from a sediment sample collected in the coast of Ubatuba – SP, using dry-stamp method (MINCER et al., 2002), in triplicate, in agar plates containing Culture Medium 2 (ROWBOTHAM; CROSS, 1977) and identified by full sequence of the 16S rDNA using the same primers as previously described for the Verrucosispora sp. isolates. Reference and experimental sequences were aligned using BioEdit version 7.2.5 with the ClustalW tool and phylogenetic analysis of full 16S rDNA sequences was conducted using the neighbor-joining algorithm (SAITOU; NEI, 1987) based on distances estimated by Kimura's two-parameter model using Molecular Evolutionary Genetics Analysis - MEGA Version 6.0 (TAMURA et al., 2013). Neighbour-joining (NJ) tree was prepared using default settings with complete deletion (FELSENSTEIN, 1985). The robustness of the resulting phylogeny was evaluated by bootstrap analysis of NJ data based on 1000 re-samplings (FELSENSTEIN, 1985). The comparison was also made by pairwise distance estimation analysis, using Kimura 2 – parameter model in MEGA6 software (TAMURA et al., 2013). Final sequence was deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) library (Pending Accession Number). 44 3.2.3.2. Fermentation, extraction and LC-HRMS analysis Strain RKMT_070 was cultivated in Marine Broth (MB – DifcoTM), at room temperature, to evaluate its capacity in the production of secondary metabolites. Thus, the following composition of media was utilized: Marine Broth (DifcoTM and BBLTM) – Peptone, 5.0 g; Yeast extract, 1.0 g; Iron citrate (III), 0.1g; NaCl, 19.45 g; MgCl, 8.8 g; Na2SO4, 3.24 g; CaCl2, 1.8 g; KCl, 0.55 g; NaHCO3, 0.16 g; KBr, 0.08 g; SrCl2, 34.0 mg; H3BO3, 22.0 mg; Na2SiO3, 4.0 mg; NaF, 2.4 mg; NH4NO3, 1.6 mg; Na2HPO4, 8.0 mg and deionized H2O, 1.0 q.s./L. The seed medium was prepared first by inoculation of a CFU (colony forming unit) of the isolate in a tube containing 7.0 mL of MB liquid medium and fermented for 3 days at room temperature. Then, 1.0 mL of the fermentation broth was inoculated in six fresh tubes containing 7.0 mL of the same liquid medium and fermented for one extra day. Finally, 120 tubes containing 7.0 mL of MB were inoculated with 210 µL (3% of broth volume) of the fermentation broth and cultivated at room temperature during 7 days at 200 rpm. Moreover two 2.0 L capacity baffled flasks containing 250 mL of MB were inoculated with 7.5 mL of the fermentation broth and cultivated in the same conditions. Tubes containing the fermentation broths were combined in a separation funnel and extracted with two portions of 500 mL of ethyl acetate. After extraction, each ethyl acetate phase was washed with two portions of 300 mL of deionized water, combined and dried. The same procedure was carried out with the fermentation broth from cultivation in baffled flasks. The remaining fermentation broth from both tubes and baffled flasks were separately extracted with HP-20 resin in a proportion of 50g/L of broth. The HP-20 was, therefore, extracted consecutively with 400 mL of H2O, MeOH/H2O 1:1 and MeOH for 30 min each and the solvent dried. All samples (ethyl acetate and HP-20 fractions from tubes and baffled flasks) were analyzed by LC-HRMS in a concentration of 500 µg mL-1 in methanol for comparison. Liquid chromatography–mass spectrometry analyses were carried out with an ESI- HRMS Thermo Scientific® EXACTIVE operating on positive mode as previously described. Compounds detected were compared to Antibase 2012 Wiley® for the identification of possible known metabolites previously reported as produced by bacteria. 45 3.2.3.3. Isolation and identification of compounds Fractions MeOH from HP-20 of the fermentation broth from tubes and baffled flasks were combined and the compounds purified using a Sunfire C18 column (5 µm, 250 × 10.00 mm, 110 Å, Waters®). Deionized H2O 0.1% formic acid (solvent A) and acetonitrile 0.1% formic acid (solvent B) were used with a flow rate of 3 mL/min and a sample concentration of 10 mg/mL. The substances were separated starting wi