P126: Linking mutations to lignocellulosic inhibitor tolerance in Saccharomyces cerevisiae

Using genome shuffling, we have evolved a strain of Saccharomyces cerevisiae (R57) that is highly tolerant to the inhibitors found in the lignocellulosic hydrolysate hardwood spent sulfite liquor (HWSSL). Many of the inhibitors found in HWSSL, including furans, phenolic compounds, organic acids as well as osmotic stress are common to various lignocellulosic hydrolysates. The genomes of both the diploid R57 mutant and haploid wildtype strains were sequenced using Illumina sequencing technology and the sequences were compared to identify the mutations accumulated in R57. We discovered and confirmed by Sanger sequencing 18 SNPs, 4 of which are homozygous and 14 of which are heterozygous. Four of these mutations are located within 205bp 5’ of the transcriptional start site and 3 are located within 42bp 3’ of the ORF. We next sought to link genotype to phenotype by identifying the mutations that are responsible for the phenotype observed. The mutated promoter/gene/terminator regions were cloned separately and expressed in the wildtype haploid strain CEN.PK 11132d. Based on their predicted functions, we separated the mutated genes onto four single copy plasmids and transformed wildtype haploid yeast with different permutations of these plasmids. Comparing the growth of these transformed cells against wildtype and R57 on HWSSL will help to elucidate which genes are most important for conferring HWSSL tolerance.