The Identification of Genetic Determinants of Methanol Tolerance in Yeast Suggests Differences in Methanol and Ethanol Toxicity Mechanisms and Candidates for Improved Methanol Tolerance Engineering

Abstract

Methanol is a promising feedstock for metabolically competent yeast strains-based biorefineries. However, methanol toxicity can limit the productivity of these bioprocesses. Therefore, the identification of genes whose expression is required for maximum methanol tolerance is important for mechanistic insights and rational genomic manipulation to obtain more robust methylotrophic yeast strains. The present chemogenomic analysis was performed with this objective based on the screening of the Euroscarf Saccharomyces cerevisiae haploid deletion mutant collection to search for susceptibility phenotypes in YPD medium supplemented with 8% (v/v) methanol, at 35 °C, compared with an equivalent ethanol concentration (5.5% (v/v)). Around 400 methanol tolerance determinants were identified, 81 showing a marked phenotype. The clustering of the identified tolerance genes indicates an enrichment of functional categories in the methanol dataset not enriched in the ethanol dataset, such as chromatin remodeling, DNA repair and fatty acid biosynthesis. Several genes involved in DNA repair (eight RAD genes), identified as specific for methanol toxicity, were previously reported as tolerance determinants for formaldehyde, a methanol detoxification pathway intermediate. This study provides new valuable information on genes and potential regulatory networks involved in overcoming methanol toxicity. This knowledge is an important starting point for the improvement of methanol tolerance in yeasts capable of catabolizing and copying with methanol concentrations present in promising bioeconomy feedstocks, including industrial residues.

The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts

“…Genes related to chitin and glucan synthesis, namely CHS1 (chitin), FKS1 (β1,3-glucans), ACF2 (β1,3-glucans), and KRE6 (β1,6-glucans), as well as genes involved in cell wall integrity ( PUN1 ) and cell wall organization ( SIT4 ), were also found to be involved in ethanol tolerance ( Kubota et al, 2004 ; Auesukaree et al, 2009 ; Teixeira et al, 2009 ; Mota et al, 2021 ). The upregulation of FKS1 and also of SED1 and SMI1 genes, involved in cell wall biosynthesis depends on the Znf1 transcription factor, recently implicated in adaptation to ethanol stress ( Samakkarn et al, 2021 ).…”

“…A recent chemogenomic analysis reported that, in S. cerevisiae , methanol and ethanol, share genetic determinants of tolerance involved in cell wall maintenance ( Mota et al, 2021 ). Methanol is a feedstock alternative to sugar-based raw substrates for biorefinery processes and a toxic compound commonly found in crude glycerol, a by-product of the biodiesel industry, and in hydrolysates from pectin-rich biomass residues ( Yasokawa et al, 2010 ; Martins et al, 2020 ; Mota et al, 2021 ). Methanol and ethanol tolerance genes include FKS1 and SMI1 (β1,3-glucan synthesis), ROT2 (β1,6-glucan synthesis), MNN11 (mannosylation of CWPs), and WSC1 (CWI pathway membrane sensor).…”

“…Methanol and ethanol tolerance genes include FKS1 and SMI1 (β1,3-glucan synthesis), ROT2 (β1,6-glucan synthesis), MNN11 (mannosylation of CWPs), and WSC1 (CWI pathway membrane sensor). However, KRE6, CWH41 (β1,6-glucan synthesis) and GAS1 (β1,3-glucan chain elongation and branching) were found to be required for maximum tolerance to methanol but not for equivalent inhibitory concentrations of ethanol ( Mota et al, 2021 ). This suggests that, despite the similarities of these alcohols, equivalent inhibitory concentrations might impact the cell wall differently and, as such, elicit not fully overlapping remodeling responses ( Mota et al, 2021 ).…”

“…However, KRE6, CWH41 (β1,6-glucan synthesis) and GAS1 (β1,3-glucan chain elongation and branching) were found to be required for maximum tolerance to methanol but not for equivalent inhibitory concentrations of ethanol ( Mota et al, 2021 ). This suggests that, despite the similarities of these alcohols, equivalent inhibitory concentrations might impact the cell wall differently and, as such, elicit not fully overlapping remodeling responses ( Mota et al, 2021 ). The CWI pathway is implicated in methanol adaptation in Komagataella phaffii (formerly Pichia pastoris ), involving the upregulation of the SLT2 homolog encoding gene in K. phaffii ( Zhang et al, 2020 ).…”

Comparative Transcriptome and Metabolome Analyses Reveal the Methanol Dissimilation Pathway of Pichia Pastoris

“…The ubiquitin-proteasome system controls almost all basic cellular processes-such as progression through the cell cycle, signal transduction, cell death, immune responses, metabolism, protein quality control and development-by degrading short-lived regulatory or structurally aberrant proteins, connecting ubiquitylation and autophagy as key regulatory events in proteasome quality control [49,50] . In Saccharomyces cerevisiae, several genes involved in DNA repair (eight RAD genes) that have been identi ed as speci c for methanol toxicity were previously reported as determinants of tolerance for formaldehyde, a methanol detoxi cation pathway intermediate [51] . Knockdown of FLD and FGH allows the accumulation of formaldehyde, leading to upregulation of DNA repair genes.…”