Screening a thermotolerant Komagataella phaffii strain with methanol cross-tolerance and the associated physiological and transcriptional responses

Komagataella phaffii is a prominent methylotrophic yeast widely used in industrial biotechnology for heterologous protein production, chemical synthesis, and C1 feedstock utilization. However, its industrial application is limited by thermal stress and methanol toxicity, while the relationship between heat adaptation and methanol tolerance remains poorly understood. Here, we obtained a thermotolerant mutant ATH through ARTP mutagenesis followed by adaptive laboratory evolution (ALE) under heat stress (37 °C). ATH tolerated heat and methanol robustly under individual stress conditions, achieving 3-fold and 8-fold higher cell densities than GS115 at 37 °C or in 5% (v/v) methanol, respectively, yet failed under the combined stress of 37 °C and 5% methanol. Using three proteins (GFP, OPH, and ChiA) as reporters, we found that ATH showed higher heterologous expression than GS115 at 33 °C. Physiologically, ATH displayed enhanced cell wall/membrane integrity, reduced ROS accumulation and lower lipid peroxidation (>30% reduction in MDA), and accelerated formaldehyde clearance. Transcriptomics identified 561 shared differentially expressed genes, with coordinated upregulation of heat shock proteins (HSP70/40/90 families; Hsp104 and Hsp70-SSA2), antioxidant enzymes (catalase, peroxiredoxin), and cell wall/membrane remodeling genes, alongside downregulation of amino acid and secondary metabolite biosynthesis. Whole-genome resequencing further distinguished ATH from GS115 by only 19 variants (9 SNPs and 10 InDels), highlighting a compact genetic basis for the evolved phenotype. This work provides a K. phaffii chassis with improved individual heat and methanol tolerance and elucidates the physiological, transcriptional, and genomic mechanism underlying heat–methanol cross-adaptation.