Construcción de cepas auxótrofas de levaduras vínicas silvestres para mejoramiento genético
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Fecha
2009
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Las levaduras de la especie Saccharomyces cerevisiae son las principales
responsables de llevar a cabo el proceso de fermentación vínica. Dentro de las diferentes
cepas de S. cerevisiae involucradas en el proceso, existen características que son
importantes desde el punto de vista enológico, como una eficiente asimilación de nitrógeno
y consumo de azúcar, una baja cantidad de azúcar residual finalizada la fermentación, así
como una alta producción de glicerol. Sin embargo, estas características no se encuentran
naturalmente en una sola cepa.
Para solucionar esta problemática, se hace uso de diferentes herramientas que
permiten obtener una cepa con dos o más características importantes para el proceso de
fermentación alcohólica. La estrategia más utilizada consiste en la generación de mutantes
auxótrofas a partir de las cepas que posean las características de interés. De esta forma, se
pueden realizar cruces entre las cepas con el objetivo de generar híbridos, los cuales deben
ser evaluados para determinar si la cepa resultante posee las dos o más características que
poseen sus cepas parentales. Es de vital importancia obtener marcadores para poder
determinar de manera rápida y fácil aquellas cepas que son híbridos, debido que una
condición que poseen las cepas nativas es ser homotálicas, y por lo tanto tienen la
capacidad de autodiploidizarse.
En esta tesis se utilizaron cassettes de disrupción génica generados por PCR para
construir cepas auxótrofas de S. cerevisiae que habían sido seleccionadas previamente por
poseer características favorables para el proceso de fermentación. De esta forma, al generar
las cepas auxótrofas, éstas podrían ser utilizadas en un programa de mejoramiento genético.
VII
Para realizarlo, se utilizó el sistema cre-loxP-kanmx. Los genes interrumpidos fueron los
genes ura3 y trp l.
Se logró reemplazar ambos alelos del gen trpl en el locus TRP 1, lo cual fue
confirmado a nivel genotípico mediante PCR. A estas cepas se les evaluó la tasa de
crecimiento, su velocidad máxima de crecimiento y su capacidad de esporulación. Los
resultados obtenidos indican que la interrupción de ambos alelos afecta significativamente
la tasa de crecimiento, la velocidad máxima de crecimiento y la capacidad de esporulación
de las cepas generadas. Por su parte, la confirmación fenotípica de los eventos de
disrupción mostró que las diferentes cepas eran capaces de crecer en medio mínimo, lo cual
indica que no se logró generar una auxotrofía para triptófano. Estos datos sugieren que las
cepas en estudio poseen copias adicionales del gen trpl en otras ubicaciones del genoma, lo
cual debe ser evaluado.
Yeast's from the species Saccharomyces cerevisiae are responsible to carry out the process of wine fermentation. In the different strains of S. cerevisiae involved in the process, there are sorne characteristics that are important for the wine process, such as efficient nitrogen assimilation and sugar consumption, a low quantity of residual sugar, so as a good production of glycerol. However, these characteristics are not found naturally in one strain. To solve this issue, different tools are used that allow obtaining one strain with two or more important characteristics for the process of alcoholic fermentation. The most used strategies consist in the generation of auxotrophic mutants from those strains that have the characteristic. In this way, crosses can be made between the strains to generate hybrids, which must be evaluated to determine if the resulting strain possesses both characteristics from the parental strains. It is extremely important to obtain markers to allow a quick and easy way to determine those strains that are hybrids, since one condition that native strains posses is that they are homothallic, and therefore have the capacity to mate with their daughter cells. In this thesis molecular tools were used to generate auxotrophic strains of S. cerevisiae that were previously selected for having favorable characteristics for the process of fermentation. In this way, the auxotrophic strains generated, could be used in a genetic improvement program. To realize the task, the cre-loxP-kanmx system was used. The interrupted genes were ura3 and trpl. Both alleles of the trpl gene located in the TRP 1 locus were replaced, which was confirmed at the genotypic level using molecular tools. In this strains the growth rate, maximum growth velocity and capacity to sporulate was measured. The results obtained indicate that the interruption in both alleles affects the growth rate and the maximum growth velocity of the strains generated. On the other hand, the phenotypic confirmation of the disruption events showed that the different strains were capable of growing in minimal medium, which indicates that they do not presentan auxotrophy. These data suggests that the strains in study possess additional copies of the trpl gene in other locations of the genome, which must be evaluated.
Yeast's from the species Saccharomyces cerevisiae are responsible to carry out the process of wine fermentation. In the different strains of S. cerevisiae involved in the process, there are sorne characteristics that are important for the wine process, such as efficient nitrogen assimilation and sugar consumption, a low quantity of residual sugar, so as a good production of glycerol. However, these characteristics are not found naturally in one strain. To solve this issue, different tools are used that allow obtaining one strain with two or more important characteristics for the process of alcoholic fermentation. The most used strategies consist in the generation of auxotrophic mutants from those strains that have the characteristic. In this way, crosses can be made between the strains to generate hybrids, which must be evaluated to determine if the resulting strain possesses both characteristics from the parental strains. It is extremely important to obtain markers to allow a quick and easy way to determine those strains that are hybrids, since one condition that native strains posses is that they are homothallic, and therefore have the capacity to mate with their daughter cells. In this thesis molecular tools were used to generate auxotrophic strains of S. cerevisiae that were previously selected for having favorable characteristics for the process of fermentation. In this way, the auxotrophic strains generated, could be used in a genetic improvement program. To realize the task, the cre-loxP-kanmx system was used. The interrupted genes were ura3 and trpl. Both alleles of the trpl gene located in the TRP 1 locus were replaced, which was confirmed at the genotypic level using molecular tools. In this strains the growth rate, maximum growth velocity and capacity to sporulate was measured. The results obtained indicate that the interruption in both alleles affects the growth rate and the maximum growth velocity of the strains generated. On the other hand, the phenotypic confirmation of the disruption events showed that the different strains were capable of growing in minimal medium, which indicates that they do not presentan auxotrophy. These data suggests that the strains in study possess additional copies of the trpl gene in other locations of the genome, which must be evaluated.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Esta tesis se realizó en el Laboratorio de Microbiología Aplicada, Departamento de Ciencia y Tecnología de los Alimentos, Facultad Tecnológica, Universidad de Santiago de Chile y fue financiada por el proyecto FONDECYT N° 1070154.
Esta tesis se realizó en el Laboratorio de Microbiología Aplicada, Departamento de Ciencia y Tecnología de los Alimentos, Facultad Tecnológica, Universidad de Santiago de Chile y fue financiada por el proyecto FONDECYT N° 1070154.
Palabras clave
Hongos, Genética