Work

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

Year: 2026

Type: article

Abstract: 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.

Source: Synthetic and Systems Biotechnology

DOI: 10.1016/j.synbio.2026.10.003

Authors: Chaoyin Li, Tianzhi Jiang, Yesong Bo, Zhenghui Lu, Yuling Zhou, Guimin Zhang

Institutions: State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei University, Beijing University of Chemical Technology

Language: English

Topic: Fungal and yeast genetics research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,500

Funders: Natural Science Foundation of Beijing Municipality, National Key Research and Development Program of China, National Key Research and Development Program of China Stem Cell and Translational Research