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However, its accumulation imposes severe stress on the microbial producer, leading to significant production losses. To address this, improving a strain\u2019s ethanol tolerance is considered an effective strategy to enhance production. In our previous research, we conducted an adaptive evolution experiment with\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    growing under gradually increasing concentrations of ethanol, which gave rise to multiple hypertolerant populations. Based on the genomic mutational data, we demonstrated in this work that adaptive alleles in the EnvZ-OmpR two-component system drive the development of ethanol tolerance in\n                    <jats:italic>E. coli<\/jats:italic>\n                    . Specifically, when a single leucine was substituted for a proline residue within the periplasmic domain using CRISPR, the mutated EnvZ osmosensor caused a significant increase in ethanol tolerance. Through promoter fusion assays, we showed that this particular mutation stabilizes EnvZ in a kinase-dominating state, which reprograms signal transduction involving its cognate OmpR response regulator. Whole-genome proteomics analysis revealed that this altered signaling pathway predominantly maintains outer membrane stability by upregulating global porin levels and attenuating ferric uptake and metabolism in the tolerant\n                    <jats:italic>envZ*<\/jats:italic>\n                    <jats:sub>L116P<\/jats:sub>\n                    mutant. Moreover, we demonstrated that the hypertolerant\n                    <jats:italic>envZ*<\/jats:italic>\n                    <jats:sub>L116P<\/jats:sub>\n                    allele also promotes ethanol productivity in fermentation, providing valuable insights for enhancing industrial ethanol production.\n                  <\/jats:p>","DOI":"10.1371\/journal.pgen.1011707","type":"journal-article","created":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T18:39:06Z","timestamp":1765996746000},"page":"e1011707","update-policy":"https:\/\/doi.org\/10.1371\/journal.pgen.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Reprogramming the EnvZ-OmpR two-component system confers ethanol tolerance in Escherichia coli by stabilizing the outer membrane and altering ferric homeostasis"],"prefix":"10.1371","volume":"21","author":[{"given":"Thomas","family":"Schalck","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9499-0946","authenticated-orcid":true,"given":"Meesha","family":"Katyal","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah","family":"De Graeve","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lars","family":"Roba","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julia Victor","family":"Baldoma","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1225-3377","authenticated-orcid":true,"given":"Toon","family":"Swings","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bram","family":"Van den Bergh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5829-0897","authenticated-orcid":true,"given":"Jan","family":"Michiels","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,12,17]]},"reference":[{"key":"pgen.1011707.ref001","volume-title":"Global warming of 1.5\u00b0C: an IPCC special report on the impacts of global warming of 1.5\u00b0C above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening 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