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Polihidroxialcanoatos (PHA) são biopolímeros naturalmente produzidos e acumulados por diversos organismos, como bactérias, archaeas e alguns eucariontes, como fungos e leveduras. São materiais termoplásticos, biodegradáveis, biocompatíveis e podem ser produzidos a partir de fontes renováveis, exibindo grande potencial para substituir plásticos produzidos a partir de recursos não renováveis. Copolímeros híbridos de PHA, que podem ser formados por monômeros de cadeia curta e média, como P(3HASCL-co-3HAMCL), apresentam características físico-químicas diferenciadas, semelhantes às dos plásticos derivados de petróleo, sendo por isso interessantes para a indústria de materiais. A PHA sintase é considerada a enzima chave na síntese de PHA, responsável por catalisar a polimerização de diferentes monômeros de (R)-hidroxiacil-CoA, influenciando a composição monomérica do polímero formado. Sistemas de recombinação baseados em transposons bacterianos são explorados como ferramentas moleculares para inserção de sequências gênicas no cromossomo de bactérias Gram-negativas. Por exemplo, elementos mini-Tn7 podem ser prontamente transferidos para a construção de cepas recombinantes. No presente trabalho, é apresentada a construção de diferentes linhagens recombinantes a partir de Pseudomonas sp. LFM 046 e LFM 461, portando em seus cromossomos genes de PHA sintase de Ralstonia eutropha, Aeromonas hydrophila ou Aeromonas sp. TSM 81. Clones candidatos foram triados quanto a inserção das sequências de interesse em seu cromossomo, sendo os positivos avaliados em relação à capacidade de produção de PHA em ensaios em agitador rotativo, com glicose como única fonte de carbono. Um dos recombinantes obtidos se mostrou produtor do copolímero P(3HB-co-3HO-co-3HD), acumulando aproximadamente 2 % de sua massa seca celular na forma de PHA. / Polyhydroxyalkanoates (PHA) are biopolymers naturally produced and accumulated by many organisms such as bacteria, archaeas and some eukaryotes, such as fungi and yeasts. As thermoplastics, biodegradable, biocompatible and possibly made from renewable resources, they exhibit great potential to replace oil-derived plastics. Hybrid PHA copolymers can be formed by short and medium-chain monomers, P(3HASCL-co-3HAMCL), and present industry desired physicochemical properties, becoming similar to conventional oil-based plastics. PHA synthase is the key enzyme in PHA biosynthesis, responsible for catalyzing the polymerization of (R)-hydroxyacyl-CoA molecules, influencing polymer monomeric composition. Tn7-based recombination strategies represent powerful molecular tolls designed for gene delivery in Gram-negative bacteria, as mini-Tn7 elements can be readily transferred to recombinants production. In this work, its presented the constructions of recombinant Pseudomonas strains harboring PHA synthase genes from Ralstonia eutropha and Aeromonas strains in specific sites of its chromosome, and the production of P(3HB-co-3HO-co-3HD). Obtained clones were screened to confirm chromosomic insertion of the phaC sequences. Positive clones PHA production and composition were evaluated in shaken-flasks assays using glucose as the only carbon source. One of the constructed recombinants accumulated P(3HB-co-3HO-co-3HD), corresponding about to 2 % of its Cell Dry Weight as PHA.

Tn7 and Tn7-like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity/fitness islands. They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins. TnsE recognizes mobile plasmids through an association with the processivity clamp and a 3′ recessed DNA end during conjugal replication. However, the mechanism for the specific recognition of 3′ recessed DNA ends remains unclear. Structural analyses of the C-terminal domain of TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. / Thesis / Master of Science (MSc)