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Previous issue date: 2017-08-08 / 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. / A crescente preocupa????o com o meio ambiente vem incentivando a busca por processos produtivos mais sustent??veis. A convers??o de substratos renov??veis em produtos qu??micos pode ser feita por s??ntese qu??mica, por??m, ?? mais comumente feita por microrganismos atrav??s de processos fermentativos. A L-ramnose (6-deoxi-L-ramnose) ?? um a????car com potencial para se tornar um qu??mico de base, j?? que pode ser usada em diversos setores industriais, como precursor para aromas artificiais e at?? como componente de cosm??ticos. Esse a????car tem um dif??cil processo de extra????o da parede celular de plantas, fato que torna sua produ????o em larga escala economicamente invi??vel. Uma fonte para L-ramnose seria a hidr??lise da mol??cula de ramnolip??deo. Rhamnolip??deos s??o biossurfactantes da classe dos glicolip??deos produzidos por Pseudomonas aeruginosa, uma bact??ria patog??nica que aumenta os custos do processo de produ????o devido a coprodu????o com toxinas. Esse surfactante tamb??m possui aplicabilidade industrial devido a sua natureza anfip??tica. Neste estudo, pela primeira vez, os genes que codificam as enzimas da via de s??ntese de dTDP-L-ramnose de P. aeruginosa a partir de glicose-1-fosfato (RmlA, RmlB, RmlC e RmlD) foram transferidos para Saccharomyces cerevisiae. Al??m disso, um gene que codifica uma sacarose fosforilase respons??vel por hidrolisar sacarose em glicose-1-fosfato e frutose, tamb??m foi inserido na levedura com a finalidade de maximizar a convers??o de sacarose a L-ramnose. Ap??s a constru????o das cepas recombinantes, apenas a atividade enzim??tica de RmlA foi detectada. No entanto, a mol??cula dTDP-L-ramnose foi detectada por espectrometria de massa. An??lises de transcri????o mostraram que h?? transcritos detect??veis para os genes codificadores de RmlA, RlmB e RmlD, mas n??o para os genes de sacarose fosforilase e RmlC. Nesse contexto, ?? prov??vel que o plasm??deo que cont??m esses genes ?? inst??vel ou que os mRNAs para esses mesmos genes sejam inst??veis, resultando na baixa produ????o das enzimas sacarose fosforilase e RmlC. Um estudo metab??lico mais detalhado das cepas deve ajudar a identificar os gargalos em torno da produ????o dessa mol??cula, facilitando o delineamento de engenharia metab??lica para aumentar sua produ????o. As cepas finais s??o base para o estabelecimento industrial da produ????o de ramnolip??deos em S. cerevisiae.
| Identifer | oai:union.ndltd.org:IBICT/oai:bdtd.ucb.br:tede/2285 |
| Date | 08 August 2017 |
| Creators | Almeida, Gabriela Carneiro de |
| Contributors | Parachin, N??dia Skorupa |
| Publisher | Universidade Cat??lica de Bras??lia, Programa Strictu Sensu em Ci??ncias Gen??micas e Biotecnologia, UCB, Brasil, Escola de Sa??de e Medicina |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UCB, instname:Universidade Católica de Brasília, instacron:UCB |
| Rights | info:eu-repo/semantics/openAccess |
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