Optimization of ethanol production in S. cerevisiae by moderate regulation of glucose metabolism genes using the CRISPR-dCas9 system

Abstract

The growing demand for renewable energy production has promoted the enhancement of the biofuels attainment industrial process. In this context, the metabolic pathways reconfiguration of the yeast S. cerevisiae - the chosen "cellular factory" for bioethanol production - appears as an alternative for productivity increase. Traditionally, the knockout or the superexpression of certain genes has been used for this purpose. To avoid the carbon flux deviation, the deletion of genes involved in glycerol synthesis, like GPD1 or GPD2, is a strategy used for enhancement of glucose fermentation. Nonetheless, gpd1-2” cells aren't effective fermenters, since glycerol synthesis is necessary for osmotic regulation and cofactors balance. Then, it is observed that gene expression regulation methods can be very advantageous, once it avoids the negative effects of binary editing. Therefore, this project proposes CRISPR-dCas9 system application based on the usage of Cas9 endonuclease without catalytic capacity fused with Mxi1 and VPR complexes to repress and activate, respectively, the expression of key genes capable of modifying ethanol productivity. The repression targets will be the GPD genes in order to obtain a decreased glycerol production without any off-target effect observed in studies of silencing these genes. Likewise, the alcohol dehydrogenase activation will be tested to stimulate an increased alcohol production. Thus, the proposal presented here makes use of audacious synthetic biology techniques for the studying of a phenomena discussed for a long time.