{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T05:08:35Z","timestamp":1783746515216,"version":"3.55.0"},"reference-count":52,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T00:00:00Z","timestamp":1767916800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22508083"],"award-info":[{"award-number":["22508083"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2022YFE0108500"],"award-info":[{"award-number":["2022YFE0108500"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>Robust yeast tolerance to inhibitors is essential for lignocellulosic biorefinery. Although cell flocculation is known to enhance acetic acid stress tolerance, the impact of its intensity remains unclear. In this study, engineered S. cerevisiae strains with distinct floc sizes were constructed through promoter engineering. The native FLO1 promoter in the non-flocculating laboratory strain BY4741 was replaced with either the constitutive strong promoter PGK1p or the ethanol-inducible promoter TPS1p using CRISPR-Cas9-mediated genome editing, resulting in strongly and moderately flocculating strains BY4741 PGK1p-FLO1 and BY4741 TPS1p-FLO1, respectively. It was revealed that the BY4741 PGK1p-FLO1 showed a survival advantage in the late-stage fermentation and severe stress condition in the presence of 7.5 g\/L acetic acid, while BY4741 TPS1p-FLO1 exhibited superior growth and fermentation performance under 5.0 g\/L acetic acid stress. Further studies suggested that the enhanced acetic acid tolerance in flocculating cells was associated with their ability to maintain significantly higher intracellular ATP levels under stress. Our work highlights the importance of optimizing flocculation properties for robust industrial fermentation, and also provides a strategic basis for engineering stress-tolerant yeast strains for efficient fermentation in inhibitor-rich cellulosic hydrolysates.<\/jats:p>","DOI":"10.3390\/jof12010047","type":"journal-article","created":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T09:25:02Z","timestamp":1767950702000},"page":"47","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Tuning FLO1 Expression via Promoter Engineering Modulates Flocculation Degree and Acetic Acid Stress Tolerance in Saccharomyces cerevisiae"],"prefix":"10.3390","volume":"12","author":[{"given":"Pei-Liang","family":"Ye","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei-Bin","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Xiong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guang-Xian","family":"Peng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheng","family":"Cheng","sequence":"additional","affiliation":[{"name":"School of Biotechnology and Food Engineering, Hefei Normal University, Hefei 230601, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin-Qing","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1007\/s13205-025-04591-0","article-title":"Recent progress on the bioconversion of lignocellulose to fuels and chemicals","volume":"15","author":"Zhu","year":"2025","journal-title":"3 Biotech"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1080\/07388551.2024.2349581","article-title":"Lignocellulosic materials valorization in second generation biorefineries: An opportunity to produce fungal biopigments","volume":"45","author":"Arruda","year":"2025","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"132605","DOI":"10.1016\/j.biortech.2025.132605","article-title":"Robust Saccharomyces cerevisiae by rational metabolic engineering for effective ethanol production from undetoxified steam-exploded corn stover hydrolysate","volume":"431","author":"Wu","year":"2025","journal-title":"Bioresour. Technol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Wang, Y., Zhang, Y., Cui, Q., Feng, Y., and Xuan, J. (2024). Composition of lignocellulose hydrolysate in different biorefinery strategies: Nutrients and inhibitors. Molecules, 29.","DOI":"10.3390\/molecules29102275"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"108562","DOI":"10.1016\/j.biotechadv.2025.108562","article-title":"Mechanism and improvement of yeast tolerance to biomass-derived inhibitors: A review","volume":"81","author":"Yu","year":"2025","journal-title":"Biotechnol. Adv."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"107947","DOI":"10.1016\/j.biotechadv.2022.107947","article-title":"Data mining of Saccharomyces cerevisiae mutants engineered for increased tolerance towards inhibitors in lignocellulosic hydrolysates","volume":"57","author":"Olsson","year":"2022","journal-title":"Biotechnol. Adv."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1007\/s00294-022-01237-z","article-title":"How adaptive laboratory evolution can boost yeast tolerance to lignocellulosic hydrolyses","volume":"68","author":"Menegon","year":"2022","journal-title":"Curr. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1039\/C4MT00275J","article-title":"Impact of zinc sulfate addition on dynamic metabolic profiling of Saccharomyces cerevisiae subjected to long term acetic acid stress treatment and identification of key metabolites involved in antioxidant effect of zinc","volume":"7","author":"Wan","year":"2015","journal-title":"Metallomics"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"217","DOI":"10.15698\/mic2023.10.806","article-title":"Investigation of the acetic acid stress response in Saccharomyces cerevisiae with mutated H3 residues","volume":"10","author":"Saha","year":"2023","journal-title":"Microb. Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"101229","DOI":"10.1016\/j.redox.2019.101229","article-title":"Redox control of yeast Sir2 activity is involved in acetic acid resistance and longevity","volume":"24","author":"Mir","year":"2019","journal-title":"Redox Biol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Cheng, C., Wang, W.B., Sun, M.L., Tang, R.Q., Bai, L., Alper, H.S., and Zhao, X.Q. (2022). Deletion of NGG1 in a recombinant Saccharomyces cerevisiae improved xylose utilization and affected transcription of genes related to amino acid metabolism. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.960114"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"126758","DOI":"10.1016\/j.biortech.2022.126758","article-title":"Manipulating cell flocculation-associated protein kinases in Saccharomyces cerevisiae enables improved stress tolerance and efficient cellulosic ethanol production","volume":"348","author":"Ye","year":"2022","journal-title":"Bioresour. Technol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1016\/j.biotechadv.2009.06.006","article-title":"Yeast flocculation: New story in fuel ethanol production","volume":"27","author":"Zhao","year":"2009","journal-title":"Biotechnol. Adv."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.jbiosc.2016.07.021","article-title":"Development of stress tolerant Saccharomyces cerevisiae strains by metabolic engineering: New aspects from cell flocculation and zinc supplementation","volume":"123","author":"Cheng","year":"2017","journal-title":"J. Biosci. Bioeng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"6908","DOI":"10.1128\/AEM.01906-14","article-title":"Flocculation causes inhibitor tolerance in Saccharomyces cerevisiae for second-generation bioethanol production","volume":"80","author":"Westman","year":"2014","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1016\/j.jbiotec.2007.07.937","article-title":"Ethanol tolerance and the variation of plasma membrane composition of yeast floc populations with different size distribution","volume":"131","author":"Lei","year":"2007","journal-title":"J. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1002\/bit.22610","article-title":"Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions","volume":"105","author":"Xue","year":"2010","journal-title":"Biotechnol. Bioeng."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Sae-Tang, K., Bumrungtham, P., Mhuantong, W., Horphul, P., Thammarongtham, C., and Laoteng, K. (2023). Engineering flocculation for improved tolerance and production of d-lactic acid in Pichia pastoris. J. Fungi, 9.","DOI":"10.3390\/jof9040409"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.apenergy.2012.03.052","article-title":"Engineering industrial Saccharomyces cerevisiae strain with the FLO1-derivative gene isolated from the flocculating yeast SPSC01 for constitutive flocculation and fuel ethanol production","volume":"100","author":"He","year":"2012","journal-title":"Appl. Energ."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1242","DOI":"10.1016\/j.bbagrm.2014.07.022","article-title":"The yeast Cyc8-Tup1 complex cooperates with Hda1p and Rpd3p histone deacetylases to robustly repress transcription of the subtelomeric FLO1 gene","volume":"1839","author":"Fleming","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1350","DOI":"10.1111\/mmi.14375","article-title":"Flocculation of Saccharomyces cerevisiae is dependent on activation of Slt2 and Rlm1 regulated by the cell wall integrity pathway","volume":"112","author":"Sariki","year":"2019","journal-title":"Mol. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"125970","DOI":"10.1016\/j.biortech.2021.125970","article-title":"High yield biorefinery products from sugarcane bagasse: Prebiotic xylooligosaccharides, cellulosic ethanol, cellulose nanofibrils and lignin nanoparticles","volume":"342","author":"Pereira","year":"2021","journal-title":"Bioresour. Technol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"6503","DOI":"10.1128\/JB.180.24.6503-6510.1998","article-title":"Region of Flo1 proteins responsible for sugar recognition","volume":"180","author":"Kobayashi","year":"1998","journal-title":"J. Bacteriol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2\u2212\u0394\u0394CT method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1016\/j.cell.2008.09.037","article-title":"FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast","volume":"135","author":"Smukalla","year":"2008","journal-title":"Cell"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1534\/g3.116.037630","article-title":"Saccharomyces cerevisiae FLO1 gene demonstrates genetic linkage to increased fermentation rate at low temperatures","volume":"7","author":"Deed","year":"2017","journal-title":"G3 Genes Genomes Genet."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"lxaf042","DOI":"10.1093\/jambio\/lxaf042","article-title":"Promoter choice for XKS1 overexpression impacts xylose metabolism in Saccharomyces cerevisiae","volume":"136","author":"Souza","year":"2025","journal-title":"J. Appl. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1186\/s13068-023-02322-2","article-title":"Genome-wide analysis reveals Hsf1 maintains high transcript abundance of target genes controlled by strong constitutive promoter in Saccharomyces cerevisiae","volume":"16","author":"Cui","year":"2023","journal-title":"Biotechnol. Biofuels Bioprod."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ymben.2011.12.003","article-title":"Ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production","volume":"14","author":"Li","year":"2012","journal-title":"Metab. Eng."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1186\/s12934-023-02223-x","article-title":"Mechanism of furfural toxicity and metabolic strategies to engineer tolerance in microbial strains","volume":"22","author":"Jilani","year":"2023","journal-title":"Microb. Cell Fact."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1007\/s00253-025-13446-w","article-title":"Engineered S. cerevisiae construction for high-gravity ethanol production and targeted metabolomics","volume":"109","author":"Yang","year":"2025","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3819","DOI":"10.3390\/foods12203819","article-title":"Simultaneous determination of 12 preservatives in pastries using gas chromatography-mass spectrometry","volume":"12","author":"Wang","year":"2023","journal-title":"Foods"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1007\/BF02931304","article-title":"Hydrogen peroxide and superoxide anion production during acetic acid-induced yeast programmed cell death","volume":"52","author":"Guaragnella","year":"2007","journal-title":"Folia Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1007\/s10295-010-0784-8","article-title":"Drug resistance marker-aided genome shuffling to improve acetic acid tolerance in Saccharomyces cerevisiae","volume":"38","author":"Zheng","year":"2011","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1007\/s00253-016-7898-5","article-title":"Overexpression of PMA1 enhances tolerance to various types of stress and constitutively activates the SAPK pathways in Saccharomyces cerevisiae","volume":"101","author":"Lee","year":"2017","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"620","DOI":"10.1002\/bit.26093","article-title":"Overexpression of OLE1 enhances stress tolerance and constitutively activates the MAPK HOG pathway in Saccharomyces cerevisiae","volume":"114","author":"Nasution","year":"2017","journal-title":"Biotechnol. Bioeng."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jbiotec.2018.12.013","article-title":"Overexpression of RCK1 improves acetic acid tolerance in Saccharomyces cerevisiae","volume":"292","author":"Oh","year":"2019","journal-title":"J. Biotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"23486","DOI":"10.1021\/acs.jafc.5c04039","article-title":"Multiple lignocellulosic inhibitor-tolerant Saccharomyces cerevisiae strains developed by evolutionary engineering and CRISPR\/Cas9 gene editing technology","volume":"73","author":"Chen","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1002\/yea.2969","article-title":"Different response to acetic acid stress in Saccharomyces cerevisiae wild-type and l-ascorbic acid-producing strains","volume":"30","author":"Martani","year":"2013","journal-title":"Yeast"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"fow010","DOI":"10.1093\/femsyr\/fow010","article-title":"Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae","volume":"16","author":"Cheng","year":"2016","journal-title":"FEMS Yeast Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1186\/s13068-019-1456-1","article-title":"Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes","volume":"12","author":"Zhang","year":"2019","journal-title":"Biotechnol. Biofuels"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1038\/45287","article-title":"The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors","volume":"402","author":"Beck","year":"1999","journal-title":"Nature"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Palma, M., Guerreiro, J.F., and S\u00e1-Correia, I. (2018). Adaptive response and tolerance to acetic acid in Saccharomyces cerevisiae and Zygosaccharomyces bailii: A physiological genomics perspective. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.00274"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4651062","DOI":"10.1155\/2019\/4651062","article-title":"Acid stress triggers resistance to acetic acid-induced regulated cell death through Hog1 activation which requires RTG2 in yeast","volume":"2019","author":"Guaragnella","year":"2019","journal-title":"Oxid. Med. Cell. Longev."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1111\/j.1574-6968.2000.tb08992.x","article-title":"Energetics of the effect of acetic acid on growth of Saccharomyces cerevisiae","volume":"184","author":"Pampulha","year":"2000","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/j.ijfoodmicro.2012.12.013","article-title":"Weak-acid preservatives: pH and proton movements in the yeast Saccharomyces cerevisiae","volume":"161","author":"Stratford","year":"2013","journal-title":"Int. J. Food Microbiol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/femsle\/fnu042","article-title":"Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance","volume":"362","author":"Ding","year":"2015","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e0041821","DOI":"10.1128\/msystems.00418-21","article-title":"A CRISPR interference screen of essential genes reveals that proteasome regulation dictates acetic acid tolerance in Saccharomyces cerevisiae","volume":"6","author":"Mukherjee","year":"2021","journal-title":"mSystems"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"RP93906","DOI":"10.7554\/eLife.93906.5","article-title":"Refining the resolution of the yeast genotype\u2013phenotype map using single-cell RNA-sequencing","volume":"13","author":"Tong","year":"2025","journal-title":"eLife"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e008492548","DOI":"10.1128\/mbio.00849-25","article-title":"A spatial transcriptomic atlas of the host response to oropharyngeal candidiasis","volume":"16","author":"Nabeela","year":"2025","journal-title":"mBio"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1239","DOI":"10.5740\/jaoacint.12-290","article-title":"Simultaneous determination of furfural, acetic acid, and 5-hydroxymethylfurfural in corncob hydrolysates using liquid chromatography with ultraviolet detection","volume":"96","author":"Dong","year":"2013","journal-title":"J. AOAC Int."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"e70244","DOI":"10.1111\/1751-7915.70244","article-title":"Chromosome III aneuploidy enhances ethanol tolerance in industrial Saccharomyces cerevisiae by increasing the TUP1 copy number","volume":"18","author":"Adam","year":"2025","journal-title":"Microb. Biotechnol."}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/1\/47\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T09:39:58Z","timestamp":1767951598000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/1\/47"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,9]]},"references-count":52,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["jof12010047"],"URL":"https:\/\/doi.org\/10.3390\/jof12010047","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,9]]}}}