Construção de cassetes de expressão lineares e transformação de Kluyveromyces marxianus para expressão de proteínas heterólogas

• Ano de defesa: 2012
• Autor(a) principal: Santos, Ancély Ferreira dos
• Orientador(a): Fietto, Luciano Gomes
• Banca de defesa: Paula, Sérgio Oliveira de , Castro, Ieso de Miranda
• 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; Proteínas heterólogas
• Palavras-chave em Inglês: Kluyveromyces marxianus; Heterologous protein
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://locus.ufv.br/handle/123456789/2442

Selection of the most suitable yeast host for heterologous protein expression is important for the success of a biotechnological process. However despite the diversity of works with protein expression in yeasts the secretion level in these organisms are still low when compared with filamentous fungi. The yeast Kluyveromyces marxianus has a great potential for fermentation and protein production. Although, there are few studies reporting heterologous expression in this yeast. It shows the need for more studies to develop an efficient system for the recombinant protein production in this yeast. The aim of this work was the construction of linear cassettes for expression heterologous proteins in K. marxianus. The cassettes were constructed by fusion PCR. For this purpose we performed a first reaction cycle in which each fragment was amplified individually (the promoter, the gene of interest and the selection mark). The promoters (ScHXT1p, ScPGKp, ScTDH3p, KmINU1p and KmPCPL3p) were amplified from the genome of S. cerevisiae and K. marxianus. The endoglucanase and celobiohidrolase genes from A. niger (eng1 and cbhA) were cloned in pKLAC1 vector that was then used as template to amplify the sequence of genes flanked by the sequences of the Kluyveromyces lactis signal peptide α-mating factor and LAC4 gene terminator. The gene KanMX, which confers resistance to geneticin (G418) in eukaryotes, was amplified from genome of a S. cerevisiae mutant. The second step was a fusion PCR reaction of the fragments: the promoter desired, α-MF-gene of interest-TT and the KanMX. This construction was efficient, reproducible and capable of being held in a shorter time than tradicional cloning methods. The constructions were used in K. marxianus UFV-3 transformation by two different methods, the lithium acetate and electroporation. The lithium acetate protocol was more efficient and the transformants were selected by resistance to increased G418 concentrations.