Identificação de QTL relacionados a resistência ao 5-(Hidroximetil) furfural em Saccharomyces cerevisiae

The need to replace fossil fuels has motivated research into renewable energy sources. In Brazil, second generation ethanol is an important alternative to fuels derived from oil. In this context, the yeast Saccharomyces cerevisiae has been demonstrated as a microorganism with commercial potential. However, aldehyde by-products with inhibitory characteristics – such as 5-(hydroxymethyl)furfural (HMF) –, derived from biomass pretreatment, are a barrier to yeasts metabolism. In this work, we evaluated the use of static growth in 96-well microplates as an alternative for the different shaking patterns used in phenotypic assays of S. cerevisiae, together with robust OD measurement correlations that overcome the low absorbance limit found in conventional plate spectrophotometers. In order to automate calculation of these physiological parameters, we created OCHT®, an open-source high-throughput phenotype profiling software for processing, analyzing and visualizing yeast growth data. Together, the cultivation standardization and data analysis toolbox allowed the evaluation and comparison of four S. cerevisiae strains (FMY001, JAY270, NCYC505 and CEN.PK-122) in media containing aldehydes growth inhibitors, typically found in hydrolysates produced from sugarcane biomass. We identified FMY001 (SA1-derived) as a tolerant strain at high concentrations of HMF, furfural and vanillin, producing biomass in presence of up to 80 mM HMF. The individual analysis of segregants derived from FMY001 revealed a haploid segregant (FMY097) with HMF tolerance similar to the parental diploid. After collecting two pools of segregants of the crossing FMY097/BY4742 with high and low tolerance to HMF, genome-wide comparison of 38820 single-nucleotide polymorphism (SNP) profiles between the two bulks allowed the identification of a region in chromosome II highly associated with the phenotype. The peak region contains 4 genes with unique non-synonymous mutations: SEA4, PKC1, SRO77 and MIX23. This study allowed us to better understand the genetic architecture of HMF resistance, which could be extended to other aldehydes. Reciprocal hemyzigosity analysis can further elucidate specific SNPs related to HMF resistance (AU)