Effects of RIM15 deletion on yeast adaptation to synthetic spruce hydrolysate.

Microbial robustness (i.e., to keep the same performance in the face of several perturbations) is a desirable trait for industrial yeasts, particularly in second-generation bioethanol production, where inhibitory compounds in lignocellulosic hydrolysates impair microbial performance. In this study, we investigated the role of the stress-responsive kinase Rim15 in stress adaptation and robustness by subjecting Saccharomyces cerevisiae wild-type (WT) and rim15Δ strain to adaptive laboratory evolution (ALE) in synthetic spruce hydrolysate (SSH). This hydrolysate contains high concentrations of inhibitory compounds, such as acetic acid and 5-hydroxymethylfurfural. Crucially, ALE consisted of two steps, including a medium swap regime that enabled adaptation to 100% SSH. Although WT and rim15Δ strain followed distinct evolutionary trajectories during ALE, both strains improved growth performance and robustness on SSH. The ability of the rim15Δ strain to adapt to 100% SSH was associated with impaired daughter cell separation and multicellular clump formation. Genome sequencing identified a mutation in a morphogenetic gene (ACE2) in Evolved rim15Δ. This differed from mutations in key transcriptional regulators (e.g., SSN8, SSN2) identified in Evolved WT. Reverse engineering indicated that ACE2 loss-of-function contributes to the multicellular clumping phenotype of the Evolved rim15Δ strain, while deletion mutants of SSN2 and SSN8 exhibited improved growth in SSH compared to the parental strain. Together, these findings indicate that yeast robustness can emerge through distinct adaptive strategies shaped by the genetic background, involving transcriptional rewiring or morphogenetic adaptation to lignocellulosic stress.