Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-14T14:43:21Z No. of bitstreams: 1 resumo.pdf: 19226 bytes, checksum: 3311f50c2162c5bb897dc91d378af6b2 (MD5) / Made available in DSpace on 2017-06-14T14:43:21Z (GMT). No. of bitstreams: 1 resumo.pdf: 19226 bytes, checksum: 3311f50c2162c5bb897dc91d378af6b2 (MD5) Previous issue date: 2005-05-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Na busca de novos resultados para elucidar o papel de HAP1 e ROX1, que codificam um ativador do metabolismo oxidativo e um repressor do metabolismo oxidorredutivo, respectivamente, na levedura Crabtree negativa Kluyveromyces lactis cuja versatilidade metabólica pode ser explorada em vários campos da biotecnologia, inicialmente, foi identificado um gene ortólogo ROX1 à S. cerevisiae na seqüência genômica de K.lactis. O gene KlROX1 possui 40% de identidade daquele presente em S. cerevisiae e um motif característico do domínio HMG (High Mobility Group). Com base nessa seqüência uma linhagem mutante com deleção de ROX1 foi construída e confirmada. O fenótipo URA + e rox1 - dos transformantes obtidos, K.lactis ∆rox1::URA3, foram 100% estáveis sob condição seletiva. O gene putativo ROX1 de K.lactis teve a sua função em resposta ao oxigênio confirmada em culturas de K. lactis sob regime contínuo e desrepressão por glicose, pois, a deleção de ROX1 induziu a um aumento no nível do transcrito do gene hipóxico HEM13. A análise dos produtos do metabolismo permitiu inferir que a deleção do gene ROX1 em K. lactis aumentou a capacidade fermentativa dessa levedura sob aerobiose e de desrepressão catabólica. A investigação em culturas K. lactis ∆hap1 submetidas ao cultivo contínuo aeróbico sob desrepressão por glicose revelou um fenótipo relacionado ao metabolismo oxidorredutivo, ou seja, K. lactis ∆hap1 é mais fermentativa levando a diversidade de metabólitos em torno do piruvato. A proposta da via de regulação parcial negativa controlando a expressão de HEM13 foi confirmada nas culturas K. lactis. / The objective of this study was to search for new results in order to elucidate the role of HAP1 and ROX1, which codify an oxidative metabolism activator and an oxidoreductive metabolism repressor respectively, in the Crabtree-negative yeast Kluyveromyces lactis, whose metabolic versatility can be exploited in several biotechnology fields. Initially, a ROX1 gene orthologous to S. cerevisiae was identified in the genomic sequence of K. lactis. The KlROX1 gene has 40% identity with the one present in S. cerevisiae and a characteristic motif of the HMG (High Mobility Group) domain. Based on this sequence, a mutant line with ROX1 deletion was built and confirmed. The obtained transformant URA + and rox1 - phenotypes, K. lactis ∆rox1::URA3, were 100% stable under selective condition. The putative K. lactis ROX1 gene had its function in response to oxygen confirmed in cultures of K. lactis under continuous regime and glucose derepression, since ROX1 deletion induced an increase in the level of the HEM13 hypoxic gene transcript. The analysis of metabolism products allowed inferring that the deletion of gene ROX1 in K. lactis increased the yeast fermentative capacity under aerobic and catabolic derepression. The investigation in K. lactis ∆hap1 cultures under continuous aerobic cultivation and glucose derepressiom revealed a phenotype related to oxidoreductive metabolism, in other words, K. lactis ∆hap1 is more fermentative, leading to metabolite diversity around the piruvate. The proposal of the partial negative regulation pathway controlling the HEM13 expression was confirmed in K. lactis cultures.

This research investigated yeast populations and diversity during pre-fermentation cold maceration and alcoholic fermentation of Vitis vinifera L. cv. Pinot noir grapes from a commercial vineyard (Dayton, OR). Fermentations were conducted at the Oregon State University research winery in 100 L tanks while grapes from the same vineyard lot were fermented at a commercial winery. Samples were taken daily during pre-fermentation maceration (9°C) and alcoholic fermentation (27°C) and plated on WL and lysine media to determine Saccharomyces and non-Saccharomyces populations and diversity. Total non-Saccharomyces populations increased from 1 x 10³ cfu/mL to 1 x 10⁵ cfu/mL during pre-fermentation cold maceration and reached a maximum of 1 x 10⁷ cfu/mL during alcoholic fermentation. Thirteen distinct yeast species were tentatively identified based on appearance on WL media and were initially screened for β-glucosidase activity using 4-methyllumbelliferyl-β-D-gluconopyranoside (4-MUG) plates. The identity of the isolates screening positive for β-glucosidase activity was determined by sequence analysis of the D1/D2 domain of the 26S rDNA gene. The five isolates identified were Metschnikowia pulcherrima, Hanseniaspora uvarum, Kluveromyces thermotolerans, and two Saccharomyces cerevisiae isolates. β-glucosidase activity was further characterized and quantified using a liquid media representing grape must conditions (pH 3.5, 20° Brix) at two temperatures (25°C and 8°C). While increasing sugar concentration suppressed the β-glucosidase activity of H. uvarum (-99%), β-glucosidase activity still remained relatively high for M. pulcherrima, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. At 8°C, β-glucosidase activity was reduced for M. pulcherrima compared to activity at 25°C, but activity increased for K. thermotolerans, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. The yeast isolates possessing β-glucosidase activity were used in fermentations of Vitis vinifera L. cv. Pinot Noir grapes. The grapes were treated with high hydrostatic pressure (HHP) to inactivate naturally occurring yeast and bacteria. All yeast isolates grew during pre-fermentation cold maceration (7 days at 9°C) and populations increased 3 to 4 logs. Following pre-fermentation cold maceration, all ferments were warmed to 27°C and inoculated with S. cerevisiae RC212. Alcoholic fermentations were all complete within eight days and after pressing wines were analyzed for volatile aroma compounds by SPME-GC-MS. The presence of different yeast isolates during pre-fermentation cold maceration resulted in wines with unique aroma profiles. Ethyl ester concentrations were highest in the wine that did not undergo a pre-fermentation cold maceration, while concentrations of branch-chained esters were higher in the treatments with yeast present during pre-fermentation cold maceration. Pre-fermentation cold maceration with yeast isolates demonstrating β-glucosidase did not affect the concentration of β-damascenone or β-ionone. Wines that had undergone pre-fermentation cold maceration with S. cerevisiae isolate 1, S. cerevisiae isolate 2, and a combination of all isolates resulted in over twice the concentration of β-citronellol over wines that did not undergo a pre-fermentation cold maceration. / Graduation date: 2013