Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris

• Ano de defesa: 2016
• Autor(a) principal: Costa, Ilítia Ganaê de Oliveira
• Orientador(a): Coelho, Alexandre Siqueira Guedes
• Banca de defesa: Coelho, Alexandre Siqueira Guedes, Silva, Silvana Petrofeza, Georg, Raphaela de Castro, Siqueira, Saulo José Linhares, Colussi, Francieli
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Goiás
• Programa de Pós-Graduação: Programa de Pós-graduação em Genética e Melhoramento de Plantas (EAEA)
• Departamento: Escola de Agronomia e Engenharia de Alimentos - EAEA (RG)
• País: Brasil
• Palavras-chave em Português: β-expansina; Saccharum officinarum; expressão heteróloga; Pichia pastoris
• Palavras-chave em Inglês: β-expansin; Saccharum officinarum; Heterologous expression; Pichia pastoris
• Área do conhecimento CNPq: GENETICA::GENETICA VEGETAL
• Link de acesso: http://repositorio.bc.ufg.br/tede/handle/tede/7365

In the last decades the search for new renewable energy sources have increased and one of the emerged technologies was the production of ethanol from lignocellulosic biomass (second generation ethanol). For obtaining fermentable sugars from biomass, a complex of enzymes and proteins that acts on polysaccharides of the plant cell wall is required. Expansins are proteins that can help enzymes in the degradation process, promoting the loosening of the cell wall of plants by breaking hydrogen bonds between cellulose fibers and hemicellulose. The superfamily of expansins has been described mainly in plants, although they are also found in fungi and bacteria. Four families are described to this superfamily: α-expansins, β-expansins, expansins-like A and expansins-like B. The α-expansins are present in most eudicots, whereas the β-expansins are involved in cellular expansion process of the monocots family Poaceae. To analyze the sequence and to verify the functional activity of a β-expansin on filter paper, in this work a gene of β-expansin (expb11) of sugarcane was isolated, cloned and expressed in the yeast Pichia pastoris. Nucleotide sequences of expansin genes from sugarcane were initially obtained from the public SUCEST database using reference sequences of expansins from sorghum, maize and rice as query. A total of 227 ESTs were identified. These ESTs were submitted to clustering and 30 contigs and 92 singletons were obtained. Specific oligos were designed and used to capture the genomic fragment corresponding to the expb11 gene by PCR. The coding DNA fragment of expb11 was obtained by RT-PCR from the total RNA extracted from stalks of the RB867515 sugarcane cultivar. This fragment was cloned into the pGEM-T Easy vector and subcloned into the pPIC9 vector. Genomic and cDNA fragments were sequenced. The expression cassette generated by subcloning into the pPIC9 expression vector was integrated into the P. pastoris strain GS115 genome, but it has not been possible to detect a functional activity of expansin. The expb11 gene comprises 1367 bp, containing 3 introns, and its cDNA is 801 bp long, with an ORF of 776 bp. The alignment of sugarcane and sorghum sequences of expb11 showed that the two species share a 95% sequence identity along these genes and that the gene structure is highly conserved in these species. The in silico analysis of the putative EXPB11 amino acid sequence allowed the detection of two conserved domains, one similar to the GH45 family, and the other to the carbohydrate binding module (CBM). The putative EXPB11 protein has a predicted molecular weight of approximately 26.4 kDa, an isoelectric point (pI) of 4.88, and contains two glycosylation sites. Our results confirmed that the isolated and cloned expb11 gene corresponds to a β-expansin gene from sugarcane, paving the way to the expression, isolation and use of recombinant EXPB11 in the mix of enzymes and proteins for lignocellulosic biomass degradation.