Development of an approach for selection of recombinant diploid based on cell cycle synchronization in Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae is an important model organism for the study of fundamental mechanisms in eukaryotes, becoming a "platform" for the production ofbiofuels. In this context, during fermentation, the yeast must be tolerant to theacetic acid present in the process, which can cause damage and even cell death. In order to obtain better and more tolerant strains, sexual evolution is a highly impacted approach. For the same to be realized, it is necessary to obtain a large number of haploids, which were initially obtained by manual dissection (a laborious method that demands time and ability of the researcher). To solve this problem, high-throughput methods were introduced using flow cytometry, such as the Barcode Enabled Sequencing ofTetrads (BEST) and the one developed by Treusch et al. However, in these methods there is still a demand for time for haploid collection, phenotyping and crossing for new phenotype analysis. This can be solved with the return to growth, in which the cells are induced to meiosis and before they pass from prophase I (point of commitment) are induced to return tothe mitosis through a nutritional shift, being able to obtain the diploid already recombined. Some of the obstacles to this methodology are the low rate of collected recombinants and the asynchrony of the cultures, where it is necessary to standardize the times of induction of sporulation for each newlineage that one wishes to use. Thus, this project aims to propose a solution for this asynchrony using the NDT80 - Gal4.ER system, in which the cells are "paused" in thepachythem. Thus, one can make a nutritional shift and a return to mitosis, obtaining directly the recombinant diploids. These recombinant diploids will be tested according to their tolerance to acetic acid, a feature of industrial interest, and will be proposed to resolve Quantitative Trait Loci (QTL) thereof, in an attempt to further resolve which genes / SNPs are responsible for this phenotype. (AU)