Expressão heteróloga de celulases em Kluyveromyces marxianus para utilização em bioprocesso consolidado de produção de etanol celulósico

• Ano de defesa: 2012
• Autor(a) principal: Rodrigues, Marina Quádrio Raposo Branco
• Orientador(a): Fietto, Luciano Gomes
• Banca de defesa: Silveira, Wendel Batista da , Oliveira, Leandro Licursi de
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Kluyveromyces marxianus; Bioprocesso; Etanol
• Palavras-chave em Inglês: Kluyveromyces marxianus; Bioprocess; Ethanol
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://locus.ufv.br/handle/123456789/2441

The high cost of cellulase is one of the main barriers for the bioconversion of lignocellulose to ethanol, and the coupling of cellulase production and the simultaneous saccharification and fermentation of bagasse, in the Consolidated Bioprocessing, can contribute to the viability of this process. This strategy requires a biocatalyst agent, or a consortium of micro-organisms, able to secrete cellulolytic enzymes and ferment the resulting sugars to ethanol. The yeast Kluyveromyces marxianus emerges in the biotechnological scenario of ethanol production by presenting key features - such as thermotolerance, production capacity of ethanol in aerated environment and high potential for protein secretion - for use in Consolidated Bioprocessing. In this work, K. marxianus UFV-3 were transformed with expression cassettes containing the cbhA or eng1 A. niger genes, which respectively encode an endoglucanase and a cellobiohydrolase, with secretion directed by the K. lactis α-MF signal peptide, under control of the S. cerevisiae PGK promoter, and the selection mark kanMX. cbhA clones were obtained with differential expression and secretion of cellobiohydrolase, in relation to non-transformed strain, suggesting that the S. cerevisiae PGK promoter, and the K. lactis α-MF signal peptide are recognized by the machinery of expression and secretion of K. marxianus UFV-3. The characterization of a cbhA clone, with increased secretion of recombinant cellobiohydrolase, suggests that the heterologous expression of recombinant proteins leads to changes in metabolic profile and secretion in the cells. The Response Surface Methodology was used to study and optimization of culture conditions that favor the secretion of endogenous and heterologous proteins in K. marxianus UFV-3. Our results indicate that the mycelial growth of K. marxianus, observed in limiting conditions of growth, is closely related to increased secretion of proteins in these conditions. The results presented here about genetic manipulation and culture conditions that promote protein secretion in K. marxianus UFV-3 open perspectives for obtaining a consortium of yeast, with recombinant and wild strains, - with ability to secrete cellulases, to hydrolyze cellulose and to ferment derived sugars - for use in Consolidated Bioprocessing on cellulosic ethanol production.