Rational bidirectional regulation of strategic genes for xylose metabolism in S. cerevisiae

Abstract

Faced with the damage caused by fossil fuels, there is a growing search for renewable fuels. Second generation ethanol (E2G), available at commercial scale in Brazil and with a low carbon footprint, is produced through the fermentation of xylose, a five-carbon sugar abundant in residual lignocellulosic biomass from the first-generation bioethanol industry. In view of this, the editing of metabolic pathways of Saccharomyces cerevisiae - the main microorganism used in industrial fermentation - is proved relevant to enable increased productivity, as this yeast is unable to ferment pentoses naturally. In addition to the insertion of genes encoding entry enzymes of the xylose consumption pathway (xylose isomerase or xylose reductase/xylitol dehydrogenase), techniques for complete deletion or repetitive insertion of certain genes have been commonly used to optimize the phenotype in recombinant strains. For example, overexpression of XKS1 or deletion of GRE3 are common editions in 2G strains, but whose trade-offs can impede optimal metabolism. In this scenario, regulation fine-tuning of gene expression is demonstrated as a viable alternative to avoid the deleterious effects of binary edits. Therefore, this project proposes the use of the CRISPR-dCas9 system to modulate the expression of target genes essential for xylose metabolism in S. cerevisiae: the overexpression target will be XKS1, and the repression target will be GRE3. Thus, different expression rates of these genes will be analyzed in an unprecedented way, using an advanced tool in synthetic biology and contributing to the construction of efficient metabolic pathways or the phenotype of interest.