Produção heteróloga de dTDP-L-ramnose em cepas recombinantes de Saccharomyces cerevisiae

• Ano de defesa: 2017
• Autor(a) principal: Almeida, Gabriela Carneiro de
• Orientador(a): Parachin, Nádia Skorupa
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Católica de Brasília
• Programa de Pós-Graduação: Programa Strictu Sensu em Ciências Genômicas e Biotecnologia
• Departamento: Escola de Saúde e Medicina
• País: Brasil
• Palavras-chave em Português: L-ramnose; Ramnolipídio; dTDP-L-ramnose; Engenharia metabólica; Saccharomyces cerevisiae
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA
• Link de acesso: https://bdtd.ucb.br:8443/jspui/handle/tede/2285

Growing concern about the environment has been encouraging the industry to seek more sustainable production processes. Although conversion of renewable substrates into chemicals can be performed through chemical reactions, it is more commonly done by microorganisms through fermentation processes. L-rhamnose (6-deoxy-L-rhamnose) is a sugar with the potential to become a chemical platform since it can be used in a range of industrial sectors as a precursor for artificial flavors and even as a cosmetic component. This sugar has a difficult plants cell walls extracting process, a fact that makes its production on a massive scale economically unfeasible. One source for L-rhamnose would be the hydrolysis of the rhamnolipid molecule. Rhamnolipids are biosurfactants of the glycolipids class produced by Pseudomonas aeruginosa, a pathogenic bacterium that increases the production process. This surfactant also has industrial applicability due to its amphipathic nature. In this study, for the first time, the genes that encodes the enzymes of the synthesis of dTDP-L-rhamnose of P. aeruginosa from glucose-1-phosphate (RmlA, RmlB, RmlC and RmlD) were transferred to Saccharomyces cerevisiae. Futhermore, a gene that encodes a sucrose phosphorylase responsible for hydrolyzing sucrose into glucose-1-phosphate and fructose was also inserted into the yeast in order to maximize the conversion of sucrose to L-rhamnose. After the recombinant strains construction, only RmlA activity was detected. However, dTDP-L-rhamnose was detected by mass spectrometry. Transcriptional analyses have shown that there are detectable transcripts for RmlA, RmlB and RmlD, but there are no detectable transcripts for sucrose – phosphorylase and RmlC. In this context, it is likely that the plasmid containing these genes is instable or that the mRNA molecules for the same genes are instable, resulting in a low expression for the enzymes for sucrose – phosphorylase and RmlC. A more detailed metabolic study of the strains should help identify bottlenecks around the production of this molecule, facilitating the metabolic engineering design to increase its production. The final strains will later be the basis of construction for the development of a S. cerevisiae rhamnolipid producing strain.