Stress-Induced Reprogramming of tRNA Epitranscriptome in the Industrially Important Yeast Komagataella phaffii

Abstract

Transfer RNA (tRNA) modifications regulate codon-biased translation and enable rapid translational adaptation to environmental stresses in diverse organisms. While these mechanisms have been extensively characterized in Saccharomyces cerevisiae, little is known about the epitranscriptomic landscape of nonconventional industrial yeasts such as Komagataella phaffii. Here, we combined comparative genomics and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) to define the tRNA epitranscriptome of K. phaffii and compare its stress-responsive remodeling to that of S. cerevisiae under oxidative and alkylating stresses. Comparative genomic analysis identified 65 shared tRNA-modifying orthologs, consistent with broad conservation of core modification machinery. LC-MS/MS profiling identified 33 modified ribonucleosides in small RNA isolated from S. cerevisiae and 35 in K. phaffii, including two modifications, lysidine (k²C) and 5,2′-O-dimethylcytidine (m⁵Cm), uniquely detected in K. phaffii. Further supporting lysidine biology in K. phaffii was the identification of a putative lysidine synthase in its genome. Exposure to equitoxic doses of methyl methanesulfonate and hydrogen peroxide induced extensive and structured remodeling of tRNA modification levels in both species. Several wobble uridine modifications associated with codon-biased translation exhibited stress- and species-dependent responses, while hierarchical clustering revealed distinct modification signatures that segregated primarily by stress type and secondarily by yeast species. Although the overall architecture of the tRNA modification system was conserved, the magnitude and direction of stress-induced remodeling differed substantially between species. These findings support a model in which conserved epitranscriptome machinery is differentially regulated to shape species-specific codon-biased translation during stress adaptation, which has implications for industrial applications of the yeast species.