Abstract
BACKGROUND: Producing second-generation ethanol from lignocellulosic hydrolysates (LCHs) poses significant challenges for Saccharomyces cerevisiae due to the presence of fermentation inhibitors. Quantitative trait loci (QTL) mapping of stress-tolerant S. cerevisiae strains is important for identifying adaptive alleles that can enhance yeast fermentation of LCHs. However, the QTL mapping process is labor-intensive, requiring the screening of numerous recombinants and repeated crossings to improve mapping resolution. RESULTS: We developed Reiterated Mass Selection and backcrosSing (ReMaSSing) to facilitate the identification of adaptive alleles through QTL mapping and to enhance LCH tolerance in yeast strains. ReMaSSing was applied to populations obtained by crossing the stress-resistant yeast PE-2_H4 with the laboratory strain S288C. Using alternative protocols, we selected haploid or diploid populations with dominant markers, enriching millions of segregants carrying adaptive alleles by propagating them in standard or LCH-supplemented media. The enriched pools were then bulk backcrossed with S288C, and germination of millions of spores generated new recombinant populations for subsequent selection cycles. After five rounds of ReMaSSing, whole-genome sequencing and QTL mapping identified key alleles associated with LCH tolerance, linked to VPS70, CAT5, GCY1, UBP2, MKT1/SAL1, HAP1, and PHO84, which influence growth and mitochondrial function in S288C. Mutations in IRA1 and HTA1, unique to our S288C strain, were also mapped, highlighting ReMaSSing's ability to "debug" the S288C background, i.e., to purge detrimental variants through selection. Allele swapping and competition assays confirmed that the identified QTL improved LCH tolerance and growth, with strains combining adaptive alleles performing over 20% better than the parental S288C. Finally, applying ReMaSSing to breed an LCH-tolerant yeast with a xylose-consuming strain produced recombinants with improved fermentation of xylose-enriched LCH. CONCLUSIONS: ReMaSSing offers a practical protocol for generating QTL mapping populations to identify adaptive alleles in tolerant strains and correct genetic defects in inferior ones. Notably, recombinant populations and clones derived from ReMaSSing outperformed both parental strains in LCH tolerance and growth. Furthermore, we applied ReMaSSing to breed strains with enhanced LCH tolerance, efficient xylose catabolism, and robust ethanol production. Together, these results demonstrate that ReMaSSing is a powerful tool for engineering industrial yeast strains that integrate desirable traits from multiple parental backgrounds.