{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T00:40:46Z","timestamp":1792456846949,"version":"4.5.0"},"reference-count":68,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T00:00:00Z","timestamp":1792454400000},"content-version":"vor","delay-in-days":89,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100000268","name":"Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["BB\/T0083690\/1"],"award-info":[{"award-number":["BB\/T0083690\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000268","name":"Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["BB\/T0083690\/1"],"award-info":[{"award-number":["BB\/T0083690\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Microb Cell Fact"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Baker\u2019s yeast,\n                      <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                      , is a widely used industrial host organism for heterologous protein production. Ensuring stable, high-level production over extended production campaigns is important for low-cost manufacturing. However, maintaining consistent yields over multiple generations is challenging as strain productivity may decline over time. Understanding potential evolutionary mechanisms underlying such decline is therefore important for optimising yeast-based expression systems in continuous manufacturing.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      In this study, diverse yeast libraries carrying high-copy-number whole-2-micron episomal expression plasmids producing a secreted SARS-CoV-2 spike protein fragment fused to mCherry were used to investigate fluorescence-based production stability during prolonged cultivation in yeast. We observed that while some strains maintained relatively stable mCherry fluorescence, most strains with high initial total fluorescence showed marked declines over 140 generations. Growth assays indicated that cultures with reduced mCherry fluorescence had a fitness advantage over high-fluorescence cultures. The decline was partly explained by phenotypic heterogeneity among genetically uniform cells; early-stage (10th generation) cultures contained distinct, non-fluorescent, low-fluorescence and high-fluorescence subpopulations (these subpopulation phenotypes were non-heritable), whereas evolved low-fluorescence cultures after 140 generations displayed loss of the high- and\/or low-fluorescence subpopulations and reduced cell-to-cell heterogeneity. Despite this, the selected evolved low-fluorescence isolates had fluorescence restored to near-initial levels after being cured of their expression plasmids and retransformed with plasmids from early-stage, high-fluorescence cultures, indicating that, in the selected evolved low-fluorescence isolates analysed, plasmid-associated changes were sufficient to explain the stable reduction in mCherry fluorescence. Whole-plasmid sequencing of the episomal 2-micron expression plasmids isolated from low-fluorescence cultures at the 140th generation revealed mutations in key functional regions, including the methionine-regulated\n                      <jats:italic>MET17<\/jats:italic>\n                      promoter driving SARS-CoV-2 protein expression,\n                      <jats:italic>FRT<\/jats:italic>\n                      recombination sites and the SARS-CoV-2 spike protein fragment coding sequence.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The results support a model in which prolonged expression of a burdensome secreted recombinant protein can be associated with early non-genetic heterogeneity in intracellular mCherry fluorescence, growth advantages of low-fluorescence cells and evolution of plasmid variants that reduce expression. The extent of fluorescence decline varied among genetically diverse strains, with some remaining comparatively stable, highlighting the potential of exploiting yeast strain diversity to obtain production strains with improved long-term stability.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12934-026-03071-1","type":"journal-article","created":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T11:27:37Z","timestamp":1784806057000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The impact of evolutionary dynamics and expression plasmid stability on long-term heterologous protein production by yeast"],"prefix":"10.1186","volume":"25","author":[{"given":"Mingzhi","family":"Xu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrei","family":"Parker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Klaus","family":"Winzer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon V.","family":"Avery","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Finnis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,23]]},"reference":[{"key":"3071_CR1","doi-asserted-by":"publisher","first-page":"379","DOI":"10.1128\/MMBR.00025-07","volume":"72","author":"E Nevoigt","year":"2008","unstructured":"Nevoigt E. Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2008;72:379\u2013412.","journal-title":"Microbiol Mol Biol Rev"},{"key":"3071_CR2","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1186\/s12934-021-01617-z","volume":"20","author":"A Madhavan","year":"2021","unstructured":"Madhavan A, Arun KB, Sindhu R, Krishnamoorthy J, Reshmy R, Sirohi R, Pugazhendi A, Awasthi MK, Szakacs G, Binod P. Customized yeast cell factories for biopharmaceuticals: from cell engineering to process scale up. Microb Cell Fact. 2021;20:124.","journal-title":"Microb Cell Fact"},{"key":"3071_CR3","doi-asserted-by":"publisher","first-page":"207","DOI":"10.4161\/bioe.22856","volume":"4","author":"J Nielsen","year":"2013","unstructured":"Nielsen J. Production of biopharmaceutical proteins by yeast: advances through metabolic engineering. Bioengineered. 2013;4:207\u201311.","journal-title":"Bioengineered"},{"key":"3071_CR4","doi-asserted-by":"publisher","first-page":"464","DOI":"10.1016\/j.tibtech.2023.09.012","volume":"42","author":"T Kjeldsen","year":"2024","unstructured":"Kjeldsen T, Andersen AS, Hubalek F, Johansson E, Kreiner FF, Schluckebier G, Kurtzhals P. Molecular engineering of insulin for recombinant expression in yeast. Trends Biotechnol. 2024;42:464\u201378.","journal-title":"Trends Biotechnol"},{"key":"3071_CR5","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1016\/j.ejbt.2019.06.003","volume":"41","author":"Q Zhang","year":"2019","unstructured":"Zhang Q, Jin Y-L, Fang Y, Zhao H. Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation. Electron J Biotechnol. 2019;41:88\u201394.","journal-title":"Electron J Biotechnol"},{"key":"3071_CR6","doi-asserted-by":"publisher","first-page":"1951","DOI":"10.2147\/IDR.S178381","volume":"12","author":"ZQ Toh","year":"2019","unstructured":"Toh ZQ, Kosasih J, Russell FM, Garland SM, Mulholland EK, Licciardi PV. Recombinant human papillomavirus nonavalent vaccine in the prevention of cancers caused by human papillomavirus. Infect Drug Resist. 2019;12:1951\u201367.","journal-title":"Infect Drug Resist"},{"key":"3071_CR7","doi-asserted-by":"publisher","first-page":"9","DOI":"10.3390\/sci6010009","volume":"6","author":"A Jayakrishnan","year":"2024","unstructured":"Jayakrishnan A, Rosli W, Tahir WR, Razak ARM, Kee FSA, Ng PE, Chew HS, Lee Y-L, Ramasamy S-K, Tan M, C. S. and, Liew KB. Evolving paradigms of pecombinant protein production in pharmaceutical industry: a rigorous review. Sci. 2024;6:9.","journal-title":"Sci"},{"key":"3071_CR8","unstructured":"Fortune Business Insights. Biopharmaceuticals market size, share & industry analysis, by type, enzymes, vaccines, hormones, by application, by distribution channel, and reginal forecast, 2024\u20132032. 2025. https:\/\/www.fortunebusinessinsights.com\/biopharmaceuticals-market-106928. Accessed 10 Apr 2025."},{"key":"3071_CR9","doi-asserted-by":"publisher","first-page":"1722","DOI":"10.1038\/s41587-022-01582-x","volume":"40","author":"G Walsh","year":"2022","unstructured":"Walsh G, Walsh E. Biopharmaceutical benchmarks 2022. Nat Biotechnol. 2022;40:1722\u201360.","journal-title":"Nat Biotechnol"},{"key":"3071_CR10","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1016\/0734-9750(95)00004-A","volume":"13","author":"BR Glick","year":"1995","unstructured":"Glick BR. Metabolic load and heterologous gene expression. Biotechnol Adv. 1995;13:247\u201361.","journal-title":"Biotechnol Adv"},{"key":"3071_CR11","doi-asserted-by":"publisher","first-page":"679665","DOI":"10.3389\/fmicb.2021.679665","volume":"12","author":"J Yang","year":"2021","unstructured":"Yang J, Tian Y, Liu H, Kan Y, Zhou Y, Wang Y, Luo Y. Harnessing the endogenous 2mu plasmid of Saccharomyces cerevisiae for pathway construction. Front Microbiol. 2021;12:679665.","journal-title":"Front Microbiol"},{"key":"3071_CR12","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1186\/s12934-018-0899-6","volume":"17","author":"L Xiong","year":"2018","unstructured":"Xiong L, Zeng Y, Tang RQ, Alper HS, Bai FW, Zhao XQ. Condition-specific promoter activities in Saccharomyces cerevisiae. Microb Cell Fact. 2018;17:58.","journal-title":"Microb Cell Fact"},{"key":"3071_CR13","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1016\/j.jbiotec.2004.04.035","volume":"113","author":"D Mattanovich","year":"2004","unstructured":"Mattanovich D, Gasser B, Hohenblum H, Sauer M. Stress in recombinant protein producing yeasts. J Biotechnol. 2004;113:121\u201335.","journal-title":"J Biotechnol"},{"key":"3071_CR14","doi-asserted-by":"publisher","first-page":"577","DOI":"10.1038\/nrmicro1460","volume":"4","author":"SV Avery","year":"2006","unstructured":"Avery SV. Microbial cell individuality and the underlying sources of heterogeneity. Nat Rev Microbiol. 2006;4:577\u201387.","journal-title":"Nat Rev Microbiol"},{"key":"3071_CR15","doi-asserted-by":"publisher","first-page":"232","DOI":"10.1007\/s43393-021-00058-4","volume":"2","author":"NX Lin","year":"2022","unstructured":"Lin NX, Xu Y, Yu XW. Overview of yeast environmental stress response pathways and the development of tolerant yeasts. Syst Microbiol Biomanufacturing. 2022;2:232\u201345.","journal-title":"Syst Microbiol Biomanufacturing"},{"key":"3071_CR16","doi-asserted-by":"publisher","first-page":"kuad033","DOI":"10.1093\/jimb\/kuad033","volume":"50","author":"X Mu","year":"2023","unstructured":"Mu X, Zhang F. Diverse mechanisms of bioproduction heterogeneity in fermentation and their control strategies. J Ind Microbiol Biotechnol. 2023;50:kuad033.","journal-title":"J Ind Microbiol Biotechnol"},{"key":"3071_CR17","doi-asserted-by":"publisher","first-page":"918","DOI":"10.1016\/j.tibtech.2022.01.004","volume":"40","author":"L Olsson","year":"2022","unstructured":"Olsson L, Rugbjerg P, Torello Pianale L, Trivellin C. Robustness: linking strain design to viable bioprocesses. Trends Biotechnol. 2022;40:918\u201331.","journal-title":"Trends Biotechnol"},{"key":"3071_CR18","doi-asserted-by":"publisher","first-page":"524","DOI":"10.1016\/j.tim.2019.01.006","volume":"27","author":"M Wehrs","year":"2019","unstructured":"Wehrs M, Tanjore D, Eng T, Lievense J, Pray TR, Mukhopadhyay A. Engineering robust production microbes for large-scale cultivation. Trends Microbiol. 2019;27:524\u201337.","journal-title":"Trends Microbiol"},{"key":"3071_CR19","doi-asserted-by":"publisher","first-page":"108401","DOI":"10.1016\/j.biotechadv.2024.108401","volume":"74","author":"J Mao","year":"2024","unstructured":"Mao J, Zhang H, Chen Y, Wei L, Liu J, Nielsen J, Chen Y, Xu N. Relieving metabolic burden to improve robustness and bioproduction by industrial microorganisms. Biotechnol Adv. 2024;74:108401.","journal-title":"Biotechnol Adv"},{"key":"3071_CR20","doi-asserted-by":"publisher","first-page":"e0162721","DOI":"10.1128\/aem.01627-21","volume":"88","author":"Y Qi","year":"2022","unstructured":"Qi Y, Xu N, Li Z, Wang J, Meng X, Gao C, Chen J, Chen W, Chen X, Liu L. Mediator engineering of Saccharomyces cerevisiae to improve multidimensional stress tolerance. Appl Environ Microbiol. 2022;88:e0162721.","journal-title":"Appl Environ Microbiol"},{"key":"3071_CR21","doi-asserted-by":"publisher","first-page":"942","DOI":"10.1021\/bp025518g","volume":"18","author":"KJ Kauffman","year":"2002","unstructured":"Kauffman KJ, Pridgen EM, Doyle FJ, Dhurjati PS, Robinson AS. Decreased protein expression and intermittent recoveries in BiP levels result from cellular stress during heterologous protein expression in Saccharomyces cerevisiae. Biotechnol Prog. 2002;18:942\u201350.","journal-title":"Biotechnol Prog"},{"key":"3071_CR22","doi-asserted-by":"publisher","first-page":"8955","DOI":"10.1007\/s00253-013-4715-2","volume":"97","author":"Z Liu","year":"2013","unstructured":"Liu Z, Hou J, Martinez JL, Petranovic D, Nielsen J. Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2013;97:8955\u201362.","journal-title":"Appl Microbiol Biotechnol"},{"key":"3071_CR23","doi-asserted-by":"publisher","first-page":"263","DOI":"10.1016\/j.ymben.2011.01.008","volume":"13","author":"A Cadiere","year":"2011","unstructured":"Cadiere A, Ortiz-Julien A, Camarasa C, Dequin S. Evolutionary engineered Saccharomyces cerevisiae wine yeast strains with increased in vivo flux through the pentose phosphate pathway. Metab Eng. 2011;13:263\u201371.","journal-title":"Metab Eng"},{"key":"3071_CR24","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1038\/nbt.3095","volume":"33","author":"K Zhou","year":"2015","unstructured":"Zhou K, Qiao KJ, Edgar S, Stephanopoulos G. Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat Biotechnol. 2015;33:377\u201383.","journal-title":"Nat Biotechnol"},{"key":"3071_CR25","doi-asserted-by":"publisher","first-page":"267","DOI":"10.1186\/s12934-024-02536-5","volume":"23","author":"M Xu","year":"2024","unstructured":"Xu M, Vallieres C, Finnis C, Winzer K, Avery SV. Exploiting phenotypic heterogeneity to improve production of glutathione by yeast. Microb Cell Fact. 2024;23:267.","journal-title":"Microb Cell Fact"},{"key":"3071_CR26","doi-asserted-by":"publisher","first-page":"947","DOI":"10.1111\/1574-6976.12073","volume":"38","author":"J Steensels","year":"2014","unstructured":"Steensels J, Snoek T, Meersman E, Picca Nicolino M, Voordeckers K, Verstrepen KJ. Improving industrial yeast strains: exploiting natural and artificial diversity. FEMS Microbiol Rev. 2014;38:947\u201395.","journal-title":"FEMS Microbiol Rev"},{"key":"3071_CR27","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1038\/nature07743","volume":"458","author":"G Liti","year":"2009","unstructured":"Liti G, Carter DM, Moses AM, Warringer J, Parts L, James SA, Davey RP, Roberts IN, Burt A, Koufopanou V, Tsai IJ, Bergman CM, Bensasson D, O\u2019Kelly MJ, van Oudenaarden A, Barton DB, Bailes E, Nguyen AN, Jones M, Quail MA, Goodhead I, Sims S, Smith F, Blomberg A, Durbin R, Louis EJ. Population genomics of domestic and wild yeasts. Nature. 2009;458:337\u201341.","journal-title":"Nature"},{"key":"3071_CR28","doi-asserted-by":"publisher","first-page":"5163","DOI":"10.1007\/s00253-020-10602-2","volume":"104","author":"SA Davison","year":"2020","unstructured":"Davison SA, den Haan R, van Zyl WH. Exploiting strain diversity and rational engineering strategies to enhance recombinant cellulase secretion by Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2020;104:5163\u201384.","journal-title":"Appl Microbiol Biotechnol"},{"key":"3071_CR29","first-page":"22Aug2024","volume":"WO2024170889","author":"C Finnis","year":"2024","unstructured":"Finnis C, Louis E, Gude C, Parker A, Smith C, Hewitt S, Kendrick E, Vallieres C, Hu Y. Yeast breeding process for strain improvement, involving PEP4 mutants. Int Patent Application. 2024;WO2024170889:22Aug2024.","journal-title":"Int Patent Application"},{"key":"3071_CR30","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1016\/S0076-6879(02)50957-5","volume":"350","author":"RD Gietz","year":"2002","unstructured":"Gietz RD, Woods RA. Transformation of yeast by lithium acetate\/single-stranded carrier DNA\/polyethylene glycol method. Methods Enzymol. 2002;350:87\u201396.","journal-title":"Methods Enzymol"},{"key":"3071_CR31","volume-title":"Current protocols in molecular biology","author":"FM Ausubel","year":"1987","unstructured":"Ausubel FM. Current protocols in molecular biology. Green Publishing, Wiley Interscience; 1987."},{"key":"3071_CR32","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1016\/S0003-2697(02)00018-0","volume":"307","author":"MV Singh","year":"2002","unstructured":"Singh MV, Weil PA. A method for plasmid purification directly from yeast. Anal Biochem. 2002;307:13\u20137.","journal-title":"Anal Biochem"},{"key":"3071_CR33","doi-asserted-by":"publisher","first-page":"953","DOI":"10.1002\/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U","volume":"14","author":"MS Longtine","year":"1998","unstructured":"Longtine MS, McKenzie A, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 1998;14:953\u201361.","journal-title":"Yeast"},{"key":"3071_CR34","doi-asserted-by":"publisher","first-page":"1065","DOI":"10.1002\/(SICI)1097-0061(19970915)13:11<1065::AID-YEA159>3.0.CO;2-K","volume":"13","author":"A Wach","year":"1997","unstructured":"Wach A, Brachat A, Alberti-Segui C, Rebischung C, Philippsen P. Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. Yeast. 1997;13:1065\u201375.","journal-title":"Yeast"},{"key":"3071_CR35","doi-asserted-by":"publisher","first-page":"1141","DOI":"10.1534\/genetics.113.155515","volume":"195","author":"FA Cubillos","year":"2013","unstructured":"Cubillos FA, Parts L, Salinas F, Bergstrom A, Scovacricchi E, Zia A, Illingworth CJ, Mustonen V, Ibstedt S, Warringer J, Louis EJ, Durbin R, Liti G. High-resolution mapping of complex traits with a four-parent advanced intercross yeast population. Genetics. 2013;195:1141\u201355.","journal-title":"Genetics"},{"key":"3071_CR36","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1002\/yea.2991","volume":"31","author":"H Louvel","year":"2014","unstructured":"Louvel H, Gillet-Markowska A, Liti G, Fischer G. A set of genetically diverged Saccharomyces cerevisiae strains with markerless deletions of multiple auxotrophic genes. Yeast. 2014;31:91\u2013101.","journal-title":"Yeast"},{"key":"3071_CR37","doi-asserted-by":"publisher","first-page":"201","DOI":"10.1016\/0378-1119(87)90376-3","volume":"58","author":"H Ma","year":"1987","unstructured":"Ma H, Kunes S, Schatz PJ, Botstein D. Plasmid construction by homologous recombination in yeast. Gene. 1987;58:201\u201316.","journal-title":"Gene"},{"key":"3071_CR38","doi-asserted-by":"publisher","first-page":"407","DOI":"10.1007\/BF00425724","volume":"200","author":"S Sangsoda","year":"1985","unstructured":"Sangsoda S, Cherest H, Surdin-Kerjan Y. The expression of the MET25 gene of Saccharomyces cerevisiae is regulated transcriptionally. Mol Genet Genomics. 1985;200:407\u201314.","journal-title":"Mol Genet Genomics"},{"key":"3071_CR39","doi-asserted-by":"publisher","first-page":"617","DOI":"10.1021\/bp049916q","volume":"21","author":"SP Solow","year":"2005","unstructured":"Solow SP, Sengbusch J, Laird MW. Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae. Biotechnol Prog. 2005;21:617\u201320.","journal-title":"Biotechnol Prog"},{"key":"3071_CR40","doi-asserted-by":"publisher","first-page":"e1001109","DOI":"10.1371\/journal.pgen.1001109","volume":"6","author":"KT Nishant","year":"2010","unstructured":"Nishant KT, Wei W, Mancera E, Argueso JL, Schlattl A, Delhomme N, Ma X, Bustamante CD, Korbel JO, Gu Z, Steinmetz LM, Alani E. The baker\u2019s yeast diploid genome is remarkably stable in vegetative growth and meiosis. PLoS Genet. 2010;6:e1001109.","journal-title":"PLoS Genet"},{"key":"3071_CR41","doi-asserted-by":"publisher","first-page":"391","DOI":"10.1007\/BF01026738","volume":"8","author":"DJ Mead","year":"1986","unstructured":"Mead DJ, Gardner DCJ, Oliver SG. Enhanced stability of a 2\u00b5-based recombinant plasmid in diploid yeast. Biotechnol Lett. 1986;8:391\u20136.","journal-title":"Biotechnol Lett"},{"key":"3071_CR42","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1007\/BF00264859","volume":"131","author":"IW Dawes","year":"1974","unstructured":"Dawes IW, Hardie ID. Selective killing of vegetative cells in sporulated yeast cultures by exposure to diethyl ether. Mol Genet Genomics. 1974;131:281\u20139.","journal-title":"Mol Genet Genomics"},{"key":"3071_CR43","doi-asserted-by":"publisher","first-page":"136","DOI":"10.1186\/1471-2164-15-136","volume":"15","author":"V Stovicek","year":"2014","unstructured":"Stovicek V, Vachova L, Begany M, Wilkinson D, Palkova Z. Global changes in gene expression associated with phenotypic switching of wild yeast. BMC Genomics. 2014;15:136.","journal-title":"BMC Genomics"},{"key":"3071_CR44","doi-asserted-by":"publisher","first-page":"345","DOI":"10.1099\/00221287-148-2-345","volume":"148","author":"ER Sumner","year":"2002","unstructured":"Sumner ER, Avery SV. Phenotypic heterogeneity: differential stress resistance among individual cells of the yeast Saccharomyces cerevisiae. Microbiology. 2002;148:345\u201351.","journal-title":"Microbiology"},{"key":"3071_CR45","doi-asserted-by":"publisher","first-page":"5802","DOI":"10.1128\/jb.176.18.5802-5813.1994","volume":"176","author":"EK Fuge","year":"1994","unstructured":"Fuge EK, Braun EL, Werner-Washburne M. Protein synthesis in long-term stationary-phase cultures of Saccharomyces cerevisiae. J Bacteriol. 1994;176:5802\u201313.","journal-title":"J Bacteriol"},{"key":"3071_CR46","doi-asserted-by":"publisher","first-page":"383","DOI":"10.1128\/mr.57.2.383-401.1993","volume":"57","author":"M Werner-Washburne","year":"1993","unstructured":"Werner-Washburne M, Braun E, Johnston GC, Singer RA. Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol reviews. 1993;57:383\u2013401.","journal-title":"Microbiol reviews"},{"key":"3071_CR47","doi-asserted-by":"publisher","first-page":"905","DOI":"10.1038\/nmeth819","volume":"2","author":"NC Shaner","year":"2005","unstructured":"Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005;2:905\u20139.","journal-title":"Nat Methods"},{"key":"3071_CR48","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1146\/annurev-cancerbio-050216-121919","volume":"1","author":"DM Fitzgerald","year":"2017","unstructured":"Fitzgerald DM, Hastings PJ, Rosenberg SM. Stress-induced mutagenesis: implications in cancer and drug resistance. Annual Rev Cancer Biology. 2017;1:119\u201340.","journal-title":"Annual Rev Cancer Biology"},{"key":"3071_CR49","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1080\/10409230701648494","volume":"42","author":"PL Foster","year":"2007","unstructured":"Foster PL. Stress-induced mutagenesis in bacteria. Crit Rev Biochem Mol Biol. 2007;42:373\u201397.","journal-title":"Crit Rev Biochem Mol Biol"},{"key":"3071_CR50","doi-asserted-by":"publisher","first-page":"221","DOI":"10.1038\/nrg3415","volume":"14","author":"RC MacLean","year":"2013","unstructured":"MacLean RC, Torres-Barcel\u00f3 C, Moxon R. Evaluating evolutionary models of stress-induced mutagenesis in bacteria. Nat Rev Genet. 2013;14:221\u20137.","journal-title":"Nat Rev Genet"},{"key":"3071_CR51","doi-asserted-by":"publisher","first-page":"399","DOI":"10.1080\/10409230701648502","volume":"42","author":"RS Galhardo","year":"2007","unstructured":"Galhardo RS, J., H. P. and and, Rosenberg SM. Mutation as a stress response and the regulation of evolvability. Crit Rev Biochem Mol Biol. 2007;42:399\u2013435.","journal-title":"Crit Rev Biochem Mol Biol"},{"key":"3071_CR52","doi-asserted-by":"publisher","first-page":"2441","DOI":"10.1093\/emboj\/16.9.2441","volume":"16","author":"L Kuras","year":"1997","unstructured":"Kuras L, Barbey R, Thomas D. Assembly of a bZIP-bHLH transcription activation complex: formation of the yeast Cbf1-Met4-Met28 complex is regulated through Met28 stimulation of Cbf1 DNA binding. EMBO J. 1997;16:2441\u201351.","journal-title":"EMBO J"},{"key":"3071_CR53","doi-asserted-by":"publisher","first-page":"00032013","DOI":"10.1128\/microbiolspec.PLAS-0003-2013","volume":"2","author":"YT Liu","year":"2014","unstructured":"Liu YT, Sau S, Ma CH, Kachroo AH, Rowley PA, Chang KM, Fan HF, Jayaram M. The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells. Microbiol Spectr. 2014;2:00032013.","journal-title":"Microbiol Spectr"},{"key":"3071_CR54","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1186\/s12934-024-02299-z","volume":"23","author":"M Zhao","year":"2024","unstructured":"Zhao M, Ma J, Zhang L, Qi H. Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae. Microb Cell Fact. 2024;23:32.","journal-title":"Microb Cell Fact"},{"key":"3071_CR55","doi-asserted-by":"publisher","first-page":"6515","DOI":"10.1038\/s41467-024-50602-8","volume":"15","author":"K Shabestary","year":"2024","unstructured":"Shabestary K, Klemm C, Carling B, Marshall J, Savigny J, Storch M, Ledesma-Amaro R. Phenotypic heterogeneity follows a growth-viability tradeoff in response to amino acid identity. Nat Commun. 2024;15:6515.","journal-title":"Nat Commun"},{"key":"3071_CR56","doi-asserted-by":"publisher","first-page":"e0230721","DOI":"10.1128\/aem.02307-21","volume":"88","author":"NR Wright","year":"2022","unstructured":"Wright NR, Jessop-Fabre MM, Sanchez BJ, Wulff T, Workman CT, Ronnest NP, Sonnenschein N. Emergence of phenotypically distinct subpopulations is a factor in adaptation of recombinant Saccharomyces cerevisiae under glucose-limited conditions. Appl Environ Microbiol. 2022;88:e0230721.","journal-title":"Appl Environ Microbiol"},{"key":"3071_CR57","doi-asserted-by":"publisher","first-page":"441","DOI":"10.1038\/s41564-022-01304-8","volume":"8","author":"S Kamrad","year":"2023","unstructured":"Kamrad S, Correia-Melo C, Szyrwiel L, Aulakh SK, Bahler J, Demichev V, Mulleder M, Ralser M. Metabolic heterogeneity and cross-feeding within isogenic yeast populations captured by DILAC. Nat Microbiol. 2023;8:441\u201354.","journal-title":"Nat Microbiol"},{"key":"3071_CR58","doi-asserted-by":"publisher","first-page":"497","DOI":"10.1038\/nrmicro3491","volume":"13","author":"M Ackermann","year":"2015","unstructured":"Ackermann M. A functional perspective on phenotypic heterogeneity in microorganisms. Nat Rev Microbiol. 2015;13:497\u2013508.","journal-title":"Nat Rev Microbiol"},{"key":"3071_CR59","doi-asserted-by":"publisher","first-page":"471","DOI":"10.1038\/ng.110","volume":"40","author":"M Acar","year":"2008","unstructured":"Acar M, Mettetal JT, van Oudenaarden A. Stochastic switching as a survival strategy in fluctuating environments. Nat Genet. 2008;40:471\u20135.","journal-title":"Nat Genet"},{"key":"3071_CR60","doi-asserted-by":"publisher","first-page":"7972","DOI":"10.1038\/ncomms8972","volume":"6","author":"D van Dijk","year":"2015","unstructured":"van Dijk D, Dhar R, Missarova AM, Espinar L, Blevins WR, Lehner B, Carey LB. Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage. Nat Commun. 2015;6:7972.","journal-title":"Nat Commun"},{"key":"3071_CR61","doi-asserted-by":"publisher","first-page":"571","DOI":"10.1038\/nature12344","volume":"500","author":"GI Lang","year":"2013","unstructured":"Lang GI, Rice DP, Hickman MJ, Sodergren E, Weinstock GM, Botstein D, Desai MM. Pervasive genetic hitchhiking and clonal interference in forty evolving yeast populations. Nature. 2013;500:571\u20134.","journal-title":"Nature"},{"key":"3071_CR62","doi-asserted-by":"publisher","first-page":"181","DOI":"10.1038\/nature14279","volume":"519","author":"SF Levy","year":"2015","unstructured":"Levy SF, Blundell JR, Venkataram S, Petrov DA, Fisher DS, Sherlock G. Quantitative evolutionary dynamics using high-resolution lineage tracking. Nature. 2015;519:181\u20136.","journal-title":"Nature"},{"key":"3071_CR63","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1186\/s12934-015-0278-5","volume":"14","author":"B Peng","year":"2015","unstructured":"Peng B, Williams TC, Henry M, Nielsen LK, Vickers CE. Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microb Cell Fact. 2015;14:91.","journal-title":"Microb Cell Fact"},{"key":"3071_CR64","first-page":"1719","volume":"12","author":"D Thomas","year":"1992","unstructured":"Thomas D, Jacquemin I, Surdin-Kerjan Y. MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae. Mol Cell Biol. 1992;12:1719\u201327.","journal-title":"Mol Cell Biol"},{"key":"3071_CR65","doi-asserted-by":"publisher","first-page":"7270","DOI":"10.1073\/pnas.82.21.7270","volume":"82","author":"JF Senecoff","year":"1985","unstructured":"Senecoff JF, Bruckner RC, Cox MM. The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. Proc Natl Acad Sci USA. 1985;82:7270\u20134.","journal-title":"Proc Natl Acad Sci USA"},{"key":"3071_CR66","doi-asserted-by":"publisher","first-page":"7380","DOI":"10.1016\/S0021-9258(17)38402-8","volume":"261","author":"JF Senecoff","year":"1986","unstructured":"Senecoff JF, Cox MM. Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing. J Biol Chem. 1986;261:7380\u20136.","journal-title":"J Biol Chem"},{"key":"3071_CR67","doi-asserted-by":"publisher","first-page":"52","DOI":"10.1016\/j.jmb.2010.07.015","volume":"402","author":"S Turan","year":"2010","unstructured":"Turan S, Kuehle J, Schambach A, Baum C, Bode J. Multiplexing RMCE: versatile extensions of the Flp-recombinase-mediated cassette-exchange technology. J Mol Biol. 2010;402:52\u201369.","journal-title":"J Mol Biol"},{"key":"3071_CR68","doi-asserted-by":"publisher","first-page":"12746","DOI":"10.1021\/bi00209a003","volume":"33","author":"T Schlake","year":"1994","unstructured":"Schlake T, Bode J. Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry. 1994;33:12746\u201351.","journal-title":"Biochemistry"}],"container-title":["Microbial Cell Factories"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12934-026-03071-1","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12934-026-03071-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12934-026-03071-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T00:02:28Z","timestamp":1792454548000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12934-026-03071-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,23]]},"references-count":68,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["3071"],"URL":"https:\/\/doi.org\/10.1186\/s12934-026-03071-1","relation":{},"ISSN":["1475-2859"],"issn-type":[{"value":"1475-2859","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,23]]},"assertion":[{"value":"20 April 2026","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"14 July 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"23 July 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"Not applicable.","order":1,"name":"Ethics","label":"Ethics approval and consent to participate","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"Not applicable.","order":2,"name":"Ethics","label":"Consent for publication","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Declarations"}}],"article-number":"218"}}