Construção de uma linhagem floculante de Kluyveromyces marxianus com potencial para produzir etanol

• Ano de defesa: 2013
• Autor(a) principal: Belo, Edgard Valdomiro Charles
• Orientador(a): Fietto, Luciano Gomes
• Banca de defesa: Queiroz, José Humberto 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 Microbiologia Agrícola
• Departamento: Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse
• País: BR
• Palavras-chave em Português: Levedura; Fermentação; Floculação; Etanol
• Palavras-chave em Inglês: Yeast; Fermentation; Flocculation; Ethanol
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::MICROBIOLOGIA AGRICOLA
• Link de acesso: http://locus.ufv.br/handle/123456789/5359

Flocculent yeasts are useful in fermentation processes because they can be easily separated from the fermentation mash reducing costs with cell separation equipment. The flocculation phenotype is not often found in native yeasts, including the species Kluyveromyces marxianus which has a high biotechnological potential due to its ability to assimilate a variety of substrates, ferment at higher temperatures and present a high growth rate. This work aimed at the construction of flocculent strains of K. marxianus with potential to ferment agro-industrial residues. The cassettes used in the construction of flocculent strains of K. marxinaus were amplified from 4 different flocculent strains of Saccharomyces cerevisiae, each strain has a cassette consisting of the URA3 gene as a selection mark and a flocculation gene FLO under control of the constitutive promoter TDH3 (URA3-TDH3p-FLO) and each of these strains contains a different flocculation gene (FLO1, FLO5, FLO9 and FLO10). In order to obtain an auxotrophic mutant strain of K. marxinaus UFV-3, the URA3 gene of this strain was amplified and cloned. Then a fragment of its coding region was removed yielding an inactive gene (URA3Δ), which was used in the transformation of K. marxinaus UFV-3. Due to the impossibility to obtain an auxotrophic strain of K. marxinaus UFV-3, we used the CBS 6556 strain auxotrophic for uracil. This strain was transformed by electroporation using the cassettes from S. cerevisiae. We obtained 4 flocculent strains of K. marxianus CBS 6556 that were selected by growth in medium without uracil. By carrying out the phenotypic analysis of the transformants, it was found that all the strains were flocculants; however, the flocculation degree was different between the 4 strains. The fermentation characteristics of the flocculent strains of K. marxianus were evaluated by using glucose, lactose and glucose/lactose as carbon and energy source and it was found that the strain transformed with the cassette containing the gene FLO9 (KMCBSFLO9) was the strain that showed the best fermentative performance preserving its ability to flocculation. In order to verify the influence of flocculation phenotype in the alcohol tolerance, the KMCBSFLO9 strain, the less flocculant strain (KMCBSFLO10) and the no-flocculant strain (KMCBSΔURA3) were cultured in the presence of different concentrations of ethanol. The results showed that the flocculent and no-flocculant strains present higher cell viability after 12 hours of culture in the presence of different concentrations of ethanol.