Whole-genome sequencing has shown that the large (8-12 Mbp) genomes of Streptomyces and allied genera of Gram-positive filamentous bacteria house a rich and previously underestimated repertoire of gene clusters for biosynthesis of specialised metabolites, including antibiotics, immunosuppressants and anticancer compounds. Many of these clusters remain uncharacterised because they are not expressed under the culture conditions used. Even for strains from which a specific compound has been identified, the challenge remains to link the compound to its gene cluster, and to develop procedures for analysing and manipulating the biosynthetic pathway. In this work, three strains have been studied that address different aspects of this challenge. Streptomyces sp. DSM4137 is a genetically amenable strain and a notably prolific producer of diverse natural products, but also has multiple biosynthetic gene clusters that remain uncharacterised. In an attempt to differentiate those clusters where the genes are expressed from those that are essentially silent, the transcriptome of DSM4137 was analysed using total RNASeq and the results were used to inform analysis of HPLC/MS data of extracts under the same conditions. There was shown to be good correlation between the RNASeq results and the pattern of metabolites produced, suggesting that RNASeq may be a useful complement in the search for novel gene clusters. In contrast, Saccharopolyspora spinosa, producer of the valuable insecticidal spinosyns, is not genetically amenable. A new technique has been developed for the mobilisation of an entire biosynthetic gene cluster and refactoring attempted to increase the production of spinosyns in a heterologous strain. Total transcriptome was analysed by RNAseq to give an insight into the regulation of the WT strain, helping identify future methods for strain manipulation for increase yields.

>Magister Scientiae - MSc / Drug-resistant infections are a global health crisis and drastically hinder the treatment options to effectively combat disease. Today, natural products remain an important source of novel drug candidates. Micro-organisms, in addition to being a source of bioactive natural products, represent a sustainable source of these compounds. As the marine environment is largely underexplored, the oceans represent a potential source of novel NPs. This study aimed at the discovery of novel NPs from bacteria associated with novel marine invertebrate species endemic to the South African coast, including a sponge Spongia (Spongia) sp. 001RSASPN and a tunicate, Pseudodistoma africanum Millar, 1954. The methodology comprised of culture-dependent and culture-independent strategies. The former involved the isolation of bacteria associated with the invertebrate species and subsequent screening for anti-microbial activity against a panel of indicator strains including a multi-drug resistant E. coli strain. Anti-bacterial activity was detected in 6.1% and 4% of bacterial isolates from the sponge and tunicate isolates respectively. The culture-independent strategy involved the use of PCR to select bioactive strains likely to contain novel NRPS or PKS secondary metabolite pathways. An NRPS A- domain exhibiting low sequence identity (65%) to reference sequences in the NCBI database was amplified from isolate PE8-15, a strain belonging to the genus Bacillus. This predicted a novel NRPS pathway within this strain. In addition, this isolate exhibited the most diverse anti-microbial profile including anti-bacterial and anti-fungal activity (A.fumigatus ATCC 46645). Therefore, as the most promising candidate, the genome of PE8-15 was sequenced following which 10 secondary metabolite pathways including bacteriocins (5), NRPS (3), siderophore (1) and a terpene pathway were identified. The A-domain amplified from PE8-15 originated from Cluster 4, and NRPS pathway predicted to encode a lipopeptide. Lipopeptides are an important class of compounds with a range of industrial applications in the pharmaceutical, cosmetic as well as food industry. The identification of potentially novel secondary metabolite pathways from even well- studied groups of organisms demonstrates the importance of sequence-based methods in natural product discovery. Furthermore, this study highlights the South African coast as a rich source of microbial natural products and should be exploited further for drug discovery.

Philosophiae Doctor - PhD / Actinobacteria, a Gram-positive phylum of bacteria found in both terrestrial and aquatic environments, are well-known producers of antibiotics and other bioactive compounds. The isolation of actinobacteria from unique environments has resulted in the discovery of new antibiotic compounds that can be used by the pharmaceutical industry. In this study, the fynbos biome was identified as one of these unique habitats due to its rich plant diversity that hosts over 8500 different plant species, including many medicinal plants. In this study two medicinal plants from the fynbos biome were identified as unique environments for the discovery of bioactive actinobacteria, Aloe ferox (Cape aloe) and Sutherlandia frutescens (cancer bush).

Magíster en Ciencias de la Ingeniería, Mención Química. Ingeniera Civil en Biotecnología / El estudios de Streptomyces, bacterias Gram positivas del filo Actinobacteria y cosmopolitas (encontradas en diferentes tipos de ambientes), ha llevado al descubrimiento de un gran número de metabolitos secundarios con potencial terapéutico. Uno de los nuevos tipos de moléculas producidas por estas bacterias son los lazo péptidos. Estos se caracterizan por formar un lazo dentro de su estructura y por presentar actividad antimicrobiana, citotóxica contra diferentes líneas celulares de cáncer y actividad inhibitoria de la replicación de ciertos virus, entre otras. Nuevos estudios han hallado cepas de Streptomyces en ambientes extremos. Una de ellas es la cepa Streptomyces sp. HST28 aislada desde el Salar de Huasco en el norte chileno. Análisis de actividad realizados a este organismo arrojaron potencial antibiótico, antifúngico y citotóxico, además su genoma fue secuenciado dando paso a la realización de minería de genomas sobre su secuencia. El presente trabajo se enfocó en la identificación de los lazo péptidos presentes en Streptomyces sp. HST28 y su caracterización. Para esto se utilizó la estrategia de minería de genomas ubicando los clústers de genes de estos péptidos, luego estos clústers fueron reconstruidos de manera bioinformática. Como segunda etapa se realizó la expresión heteróloga de cada lazo péptido empleando como huésped Streptomyces coelicolor M1152 y M1154. Una vez clonados se detectó el péptido a través de espectrometría de masa y se evaluó su la actividad. Paralelamente al estudio de laboratorio se realizó una investigación in silico de cada lazo péptido creando modelos por homología de secuencia y estructurales, usando como plantilla o molde lazo péptidos ya descritos y caracterizados estructuralmente, buscando similitudes en campos electrostáticos, distribuciones de hidrofobicidad y espaciales. Se encontraron cuatro clústers de genes de lazo péptidos en el genoma de Streptomyces sp. HST28, nombrados como LP1, LP2, LP3 y LP4. Tres de estos lazo péptidos fueron clasificados como clase II y uno de clase I (LP1). La organización de sus clústers es acorde a lo encontrado en bibliografía a excepción del de LP3. Las secuencias codificantes para los cuatro péptidos se clonaron en el vector pIJ10257, que posee el promotor constitutivo ermE*. Solo LP1 y LP3 fueron clonados exitosamente en S. colelicolor M1152, el producto predicho para LP1 no fue encontrado en los clones analizados por espectrometría de masa. En la investigación in silico se logró modelar los cuatro lazo péptidos y se realizó una comparación preliminar con otras estructuras, deduciendo que es posible implementar esta metodología para tener una aproximación del comportamiento de estas moléculas.

Os compostos de origem microbiana tem readquirido interesse pela biodisponibilidade, especificidade de alvo e diversidade química, mas as vias biosintéticas permanecem crípticas em condições de cultura. Uma estratégia para expressa-las é a super-expressão de genes ativadores. O laboratório de Bio-Produtos no ICB na USP tem trabalhado com Streptomyces olindensis produtor da Cosmomicina D uma molécula com atividade antitumoral de interesse devido ao padrão de glicosilação. O genoma de S. olindensis foi sequenciado e submetido ao NCBI (JJOH00000000) e utilizando o software antiSMASH foram identificados 33 clusters envolvidos na produção de metabolitos secundários. Encontraram-se clusters gênicos para a produção de metabolitos como Melanina, Geosmina, entre outros. Além, foi realizada uma analise de genômica comparativa para caracterizar e anotar as 22 vias biossintéticas desconhecidas em S. olindensis. Finalmente, escolheram-se a via do aminociclitol e um Policetídeo Tipo I para a super-expressão de genes reguladores levando a detecção do composto sob condições de cultura. / Microbial metabolites regain interest due to its bioavailability, target specificity and chemical diversity, but the biosynthetic pathways remain silenced under culture conditions. A strategy to obtain them is the over expression of regulatory genes. Bio-products laboratory at USP has been working with Streptomyces olindensis, products of Cosmomycin D, an antitumoral molecule with a distinctive glycosylation pattern. S. olindensis genome was sequenced and submitted to NCBI (JJOH00000000) and employing antiSMASH server 33 secondary metabolite related clusters were identified. Known pathways were found such as genes for melanin production, Geosmin and others. Additionally, a comparative genomic approach was used to characterize the 22 biosynthetic unknown pathways described in S. olindensis. Subsequently, Aminocyclitol and Polyketide Type I were chosen to evaluated, over expressing the regulatory genes, leading to the compound detection in regular culture conditions.

Os compostos de origem microbiana tem readquirido interesse pela biodisponibilidade, especificidade de alvo e diversidade química, mas as vias biosintéticas permanecem crípticas em condições de cultura. Uma estratégia para expressa-las é a super-expressão de genes ativadores. O laboratório de Bio-Produtos no ICB na USP tem trabalhado com Streptomyces olindensis produtor da Cosmomicina D uma molécula com atividade antitumoral de interesse devido ao padrão de glicosilação. O genoma de S. olindensis foi sequenciado e submetido ao NCBI (JJOH00000000) e utilizando o software antiSMASH foram identificados 33 clusters envolvidos na produção de metabolitos secundários. Encontraram-se clusters gênicos para a produção de metabolitos como Melanina, Geosmina, entre outros. Além, foi realizada uma analise de genômica comparativa para caracterizar e anotar as 22 vias biossintéticas desconhecidas em S. olindensis. Finalmente, escolheram-se a via do aminociclitol e um Policetídeo Tipo I para a super-expressão de genes reguladores levando a detecção do composto sob condições de cultura. / Microbial metabolites regain interest due to its bioavailability, target specificity and chemical diversity, but the biosynthetic pathways remain silenced under culture conditions. A strategy to obtain them is the over expression of regulatory genes. Bio-products laboratory at USP has been working with Streptomyces olindensis, products of Cosmomycin D, an antitumoral molecule with a distinctive glycosylation pattern. S. olindensis genome was sequenced and submitted to NCBI (JJOH00000000) and employing antiSMASH server 33 secondary metabolite related clusters were identified. Known pathways were found such as genes for melanin production, Geosmin and others. Additionally, a comparative genomic approach was used to characterize the 22 biosynthetic unknown pathways described in S. olindensis. Subsequently, Aminocyclitol and Polyketide Type I were chosen to evaluated, over expressing the regulatory genes, leading to the compound detection in regular culture conditions.

Biotin is an essential vitamin in plants and mammals functioning as the carbon dioxide carrier within central lipid metabolism. Biotin is composed of a fused bicylic ring system and a five carbon, carboxylic acid chain. Biotin biosynthesis in bacteria is catalysed by a series of enzymes that use fatty acid, amino acid and sulfur-containing substrates. In Bacillus subtilis, pimeloyl-CoA synthetase (PCAS, EC 6.2.1.14, UNIPROT code: P53559, 29.6 kDa) is the first enzyme in the biotin biosynthetic pathway and acts as a highly specific substrate selection gate ensuring the integrity of the carbon chain in biotin synthesis. PCAS catalyses the synthesis of the key acyl-thioester, pimeloyl-CoA in two steps; the first involves activation of pimelic acid (C7 dicarboxylic acid) using ATP to give an acyl-adenylate, enzyme-bound intermediate and pyrophosphate (PPi), and in the second step, this pimeloyl-adenylate reacts with coenzyme A (CoASH) to form the pimeloyl-CoA thioester. This thesis describes the results of biochemical, structural and mechanistic studies of B. subtilis PCAS. Recombinant PCAS was prepared by expressing the B. subtilis BioW gene in E. coli in various hexa-histidine affinity-tagged forms and the enzyme purified in high purity and yield. Enzyme activity and kinetic constants were measured using reverse-phase HPLC and enzyme coupled spectroscopic assays. These revealed the enzyme to have a strict carboxylic acid specificity. In collaboration with colleagues at the University of St. Andrews various commercial and in-house screens were used to obtain diffraction-quality crystals suitable for X-ray crystallography. This also included the generation of seleno-methionine (SeMet) labelled PCAS, as well as heavy-metal derivatives. Structures of B. subtilis PCAS in complex with the substrate pimelic acid and the pimeloyl-adenylate intermediate and product PPi were determined at 2.04 Å and 2.34 Å resolution respectively. The B. subtilis PCAS displays a novel 3D fold and defines a new class (Class IV) in the ANL superfamily of adenylate forming enzymes. The enzyme is a homodimer composed of two domains, a short N-terminus and a large C-terminal domain and the ligand-bound structures revealed the residues potentially involved in substrate specificity and enzyme catalysis. The enzyme uses an internal ruler composed of a number of conserved arginine residues (Arg213, Arg227 and Arg170) to select the correct dicarboxylic acid substrate. The X-ray structures guided the production of a number of site directed mutants to identify residues involved in the catalytic mechanism and stabilising the acyl-adenylate intermediate. This also allowed rational engineering of the PCAS active site to generate mutants with altered substrate specificity. Mutant PCAS Y211F was shown to synthesise both heptanoyl (C7) and octanoyl (C8) mono carboxylic acid-CoA and C8 dicarboxylic-CoA thioester products, highlighting the synthetic potential of PCAS. The PCAS pimeloyl-CoA product is the substrate for the next enzyme in the biotin pathway, a pyridoxal 5'phosphate (PLP)-dependent 8-amino 7-oxononanoate synthase (AONS). AONS catalyses the condensation of pimeloyl-CoA with L-alanine to give AON which is converted to biotin by the action of three other enzymes. We used genome mining to identify a putative ~66 kDa, bi-functional PCAS/AONS enzyme with an N-terminal PCAS domain fused to C-terminal AONS domain in the organism Corynebacterium amycolatum. A recombinant C. amycolatum PCAS/AONS fusion protein was expressed and purified from E. coli and initial studies suggest that it forms a functional, fused, dimeric enzyme.

>Magister Scientiae - MSc / The continued emergence of bacterial resistance to the antibiotics currently employed to treat several diseases has added to the urgency to discover and develop novel antibiotics. It is well established that natural products have been the source of the most effective antibiotics that are currently being used to treat infectious diseases and they remain a major source for drug production. Natural products derived from marine microorganisms have received much attention in recent years due to their applications in human health. One of the biggest bottlenecks in the drug discovery pipeline is the rediscovery of known compounds. Hence, dereplication strategies such as genome sequencing, genome mining and LCMS/MS among others, are essential for unlocking novel chemistry as it directs compound discovery away from previously described compounds. In this study, the genome of a marine microorganism, Thalassomonas viridans XOM25T was mined and its antimicrobial activity was assessed against a range of microorganisms. Genome sequencing data revealed that T. viridans is a novel bacterium with an average nucleotide identity of 81% to its closest relative T. actiniarum. Furthermore, genome mining data revealed that 20% of the genome was committed to secondary metabolisms and that the pathways were highly novel at a sequence level. To our knowledge, this species has not previously been exploited for its antimicrobial activity. Hence, the aim of this study was to screen for bioactivity and identify the biosynthetic gene/s responsible for the observed bioactivity in T. viridans using a bioassay-and-genome- guided isolation approach to assess the bioactive agent. The bioassay-guided fractionation approach coupled to LCMS/MS led to the identification of a novel antimicrobial protein, TVP1. Bioinformatic analyses showed that TVP1 is a novel antimicrobial protein that is found in the tail region of a prophage in the T. viridans genome. Phage-derived proteins have previously been shown to induce larval settlement in some marine invertebrates. Since the mechanism of action of TVP1 remains unknown, it remains a speculation whether it may offer a similar function. More research is required to determine the biotechnological application and the role of TVP1 in its host and natural environment.

Orientador: Luciana Gonzaga de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T08:47:55Z (GMT). No. of bitstreams: 1 Cruz_PedroLuisRochada_M.pdf: 2656746 bytes, checksum: 52eb866ff62a51e20e65bc5b01101aeb (MD5) Previous issue date: 2011 / Resumo: Actinobactérias são fontes importantes para a descoberta de novas moléculas com atividades biológicas destacadas. A este grupo pertencem Streptomyces, micro-organismos que possuem dentre outros, dois grupos de enzimas multimodulares conhecidas como policetídeo sintase (PKS) e peptídeo não ribossomal sintetase (NRPS) que catalisam a produção de policetídeos e peptídeos não ribossomais respectivamente. A biossíntese destas moléculas se dá geralmente seguindo uma relação linear entre o gene, a enzima e a estrutura da molécula. Desta forma, com o conhecimento das sequências do gene é possível prever a molécula a ser produzida e sua manipulação amplia a possibilidade de obter novas moléculas. Neste sentido foi adotada uma estratégia para o mapeamento dos genes biossintéticos sem a necessidade do sequenciamento extensivo do micro-organismo com o objetivo de prever o tipo de policetídeo e peptídeo não ribossomal produzido por linhagens de Streptomyces isoladas de Citrus ssp. Com o objetivo de conhecer os metabólitos produzidos por estes micro-organismos foi feito também o cultivo destes em meios contendo fontes de nutrientes variadas e o monitoramento de metabólitos presentes nos extratos utilizando técnicas de cromatografia líquida acoplada com espectrometria de massas. Ensaios em placas permitiram a visualização da produção de sideróforos e a atividade antibiótica dos meios de cultivo / Abstract: Actinobacteria are important sources of new molecules with biological activities. Streptomyces are the most important genera studied. Such microorganisms carries out among a variety of biosynthetic enzimes, two main groups known as polyketide synthase (PKS) and non ribosomal peptide synthetase (NRPS), both catalyzing the biosynthesis of polyketide and non ribosomal peptides respectively. PKS and NRPS biosynthesis follows a collinear relationship among the gene cluster, the enzyme and the secondary metabolite. Therefore, the knowledge of the gene sequences allows to find new molecules. In this work we adopted a strategy to map the biosynthetic genes without the need of extensive whole genome sequencing in order to predict the metabolite produced by Streptomyces strains isolated from Citrus ssp. The strains were also cultivated and the metabolite production monitored by liquid chromatography coupled with mass spectrometry. In addition, the biological activity of these medium was estimated using qualitative biochemical assays / Mestrado / Quimica Organica / Mestre em Química

Doctor en Ciencias de la Ingeniería, Mención Ingeniería Química y Biotecnología / Streptomyces leeuwenhoekii C34 es un actinomiceto aislado del desierto de Atacama, Chile, productor de los antibióticos chaxamicinas A a D y chaxalactinas A a C. El objetivo de este trabajo fue identificar los genes involucrados en la biosíntesis de chaxamicinas y chaxalactinas y producir chaxamicinas en un huésped heterólogo. Capítulo Uno detalla procedimientos de crecimiento y modificación genética de S. leeu- wenhoekii C34. La temperatura óptima para producción de chaxamicinas en medio líquido fue 30 °C, y la que permitió alcanzar altos niveles de esporulación en medio sólido fue 37 °C. S. leeuwenhoekii C34 fue sensible a tioestreptona, apramicina, higromicina B y kanamicina, mientras que no a ácido nalidíxico, antibióticos usados para seleccionar bac- terias genéticamente modificadas. Un vector sensible a temperatura (pGM1190) y otro con un sistema de integración del fago C31 (pSET152) fueron exitosamente transferi- dos a S. leeuwenhoekii C34 por conjugación con una cepa que no metila ADN, E. coli ET12567/pUZ8002. Suplementación del medio de conjugación con 120 mM MgCl2 ó 60 mM CaCl2, incrementó la frecuencia de conjugación de forma significativa, comparado con el control sin adición de sales. En este trabajo se demostró que S. leeuwenhoekii C34 incorpora ADN foráneo no metilado. Capítulo Dos describe la identificación y expresión heteróloga de los genes de biosín- tesis de chaxamicinas. El genoma de S. leeuwenhoekii C34 fue secuenciado de novo combinando tecnologías de secuenciación Illumina y PacBio. Un grupo de 27 genes (80,2 kb, locus 1.210.347 1.290.550), codifica enzimas para la biosíntesis de chaxam- icinas. pIJ12853 contenía un inserto del genoma de S. leeuwenhoekii C34 de 145 kb que incluye el segmento de 80,2 kb, el que fue transferido a una cepa que no produce chaxamicinas, S. coelicolor M1152, resultando en la producción de chaxamicinas A D, confirmando que los genes presentes en pIJ12853 codifican para la ruta de biosíntesis de chaxamicinas. Genes del huésped heterólogo podrían estar involucrados en biosíntesis y/o exporte de estas moléculas. La deleción del gen AHBA sintasa (cxmK) en S. leeu- wenhoekii C34, dio origen a la cepa M1653 que pierde su capacidad de producir chax- amicinas. Para demostrar que la interrupción en la producción de chaxamicinas fue sólo debido a su incapacidad de producir AHBA, un cultivo líquido de M1653 suplementado con AHBA comercial permitió restablecer la producción de chaxamicinas. Esto es una prueba más de que el los genes de biosíntesis de chaxamicinas fueron identificados. Capítulo Tres describe la identificación bioinformática de los genes de biosíntesis de chaxalactinas. Una secuencia de 80,7 kb (locus 7.146.903 7.227.608) contenía genes codificantes de 5 subunidades de una policétido sintasa, cuya arquitectura de dominios coincidía con la predicha para biosíntesis de chaxalactina A. Un gen que codifica una citocromo P450 sería responsable de la hidroxilación C-14 de chaxalactina A que da ori- gen a chaxalactina B. Dos genes codificantes de O-metiltransferasas estarían involucra- dos en una O-metilación C-13 de chaxalactina B, que da origen a chaxalactina C.