Análise da variação cariotípica e dos mecanismos de recombinação em leveduras industriais (Saccharomyces cerevisiae) durante o processo de fermentação alcoólica / Analysis of the karyotypic variation and the recombination mechanisms industrial yeast (Saccharomyces cerevisiae) during the alcoholic fermentation process

Fabiana de Melo Duarte

Brazilian sugar cane ethanol stands out among the energetic alternatives available nowadays. In the fermentation process to produce ethanol it is used the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is the industrial isolate PE-2, currently used by ~30% of Brazilian distilleries, generating ~10% of the worlds ethanol supply. This strain combines a high performance in ethanol fermentation and an excellent adaptation to the hostile environment in the fermentation vats. Our research group previously carried out a thorough genetic characterization of an isolate of the PE-2 strain, known as JAY270. This analysis offers several opportunities for the improvement and modification of this strain. However, some studies observed the occurrence of karyotypic variation in this strain during the fermentation process, what can represent a barrier to the genetic manipulation of this yeast. Therefore, it is extremely important to study the genome behavior of this yeast during the ethanol production process. The main objective of this Masters Project was to determinate the genetic recombination mechanism responsible for generating chromosomal rearrangements in JAY270 during the ethanol production. A fermentation experiment in semi-industrial scale was carried out with a pure initial inoculum of JAY270. After 50 days, samples were collected and used to obtain single colony isolates. This isolates were analyzed through PFGE (Pulsed Field Gel Electrophoresis) to the identification of JAY270 derivatives carrying karyotypic variation. Of the analyzed derivatives, 36% had at least one chromosomal rearrangement. 11 of these derivatives were chosen to perform detailed analyses and were called FDY1-FDY11. The genome sequences of two haploid derivatives of JAY270 were used to develop 9 molecular markers to genotype heterozygous regions of JAY270 to identify genetic recombination events. The 11 isolates were analyzed with these markers and, except by one of them, all isolates were heterozygous in all genotyped regions. Two genetic recombination mechanisms can be responsible for generating the rearrangements, mitotic recombination, which occurs in isolate points of the genome, and meiotic recombination, that simultaneously affects all the genome in one single cellular cycle. The probability of the 10 isolates stay heterozygous in all genotyped regions after one event of meiosis followed by mating is extremely low (between 10-24 e 10-14). This result showed that these rearrangements couldnt be generated through meiotic recombination. Therefore, it was possible to conclude that the chromosomal rearrangements were generated during vegetative growth through mitotic recombination. The 11 isolates also were analyzed through CGH-array to compare their gene dosage with the strain JAY270. With this methodology it was possible to detect mitotic recombination events that resulted in loss of heterozygosity in peripheral regions of some chromosomes.

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