{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T11:33:32Z","timestamp":1786707212960,"version":"build-2736575974"},"reference-count":43,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100008766","name":"Science and Technology Innovation Special Fund Project of Fujian Agriculture and Forestry University","doi-asserted-by":"publisher","award":["KFB23075A"],"award-info":[{"award-number":["KFB23075A"]}],"id":[{"id":"10.13039\/501100008766","id-type":"DOI","asserted-by":"publisher"}]},{"award":["KFB23075A"],"award-info":[{"award-number":["KFB23075A"]}],"id":[{"id":"https:\/\/ror.org\/04kx2sy84","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J. Fungi","JoF"],"abstract":"<jats:p>Aspergillus flavus, a ubiquitous phytopathogen, produces mycotoxins, especially aflatoxin B1 (AFB1), and infects crops worldwide. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the glycerol synthesis and metabolic pathway catalyzing the reversible conversion reaction between glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). However, the biological function of G3PDH in A. flavus remains uncharacterized. In this study, the glycerol-3-phosphate dehydrogenase GfdA and GfdB recombinant proteins of A. flavus were expressed, and the enzymatic activity of the GfdA protein was successfully determined. Subsequently, single-gene knockout strains (\u0394gfdA, \u0394gfdB), double-gene knockout strains (\u0394gfdA\u0394gfdB) and their corresponding complemented strains (gfdAC, gfdBC) were constructed by a homologous recombination method to explore the biological functions of these two genes in A. flavus. The phenotypic analyses revealed that although both gfdA and gfdB encoded glycerol-3-phosphate dehydrogenases, gfdA plays major roles in colony growth, conidiation, sclerotium formation, crop infection and osmotic stress tolerance in A. flavus. Notably, the performance of the \u0394gfdA\u0394gfdB strains is almost similar to that of the \u0394gfdA strain. Biochemical assays demonstrated that the intracellular glycerol content increased significantly in all mutants compared to the wild type (WT) under both normal and osmotic stress conditions. Furthermore, we found that exogenous glycerol supplementation rescued the growth defect of the \u0394gfdA and \u0394gfdA\u0394gfdB strains. Taken together, GfdA is important for glycerol synthesis, while GfdB is functionally redundant with respect to GfdA. This study preliminarily explores the main biological functions of GfdA and GfdB, providing a theoretical basis for the study of glycerol anabolic pathways of A. flavus and also offering novel insights into the development of strategies to control aflatoxin contamination.<\/jats:p>","DOI":"10.3390\/jof12080610","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T10:39:01Z","timestamp":1786703941000},"page":"610","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Role of Glycerol-3-Phosphate Dehydrogenase in the Development, Pathogenicity, and Glycerol Biosynthesis of Aspergillus flavus"],"prefix":"10.3390","volume":"12","author":[{"given":"Liurong","family":"Zhang","sequence":"first","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaru","family":"Zhao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"},{"name":"School of Biological Engineering, Jiao Zuo College of Industry and Trade, Jiaozuo 454550, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-4711-7633","authenticated-orcid":false,"given":"Hongyi","family":"Lin","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaoze","family":"Wu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Wu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongtai","family":"Ning","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuna","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Yuan","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanling","family":"Yang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.fct.2018.11.047","article-title":"Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods","volume":"124","author":"Rushing","year":"2019","journal-title":"Food Chem. Toxicol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"740","DOI":"10.3109\/10408444.2011.575766","article-title":"Aflatoxins and growth impairment: A review","volume":"41","author":"Khlangwiset","year":"2011","journal-title":"Crit. Rev. Toxicol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1677","DOI":"10.1099\/mic.0.2007\/007641-0","article-title":"Aspergillus flavus: Human pathogen, allergen and mycotoxin producer","volume":"153","author":"Hedayati","year":"2007","journal-title":"Microbiology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2021","DOI":"10.1007\/s10096-019-03636-6","article-title":"Could aflatoxin B1 production by affect the severity of keratitis: An experience in two tertiary health care centers, Egypt","volume":"38","author":"Barakat","year":"2019","journal-title":"Eur. J. Clin. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Benkerroum, N. (2019). Retrospective and Prospective Look at Aflatoxin Research and Development from a Practical Standpoint. Int. J. Environ. Res. Public Health, 16.","DOI":"10.20944\/preprints201908.0218.v1"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Pickova, D., Ostry, V., and Malir, F. (2021). A Recent Overview of Producers and Important Dietary Sources of Aflatoxins. Toxins, 13.","DOI":"10.3390\/toxins13030186"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1007\/s12550-016-0265-7","article-title":"Mycotoxins as human carcinogens-the classification","volume":"33","author":"Ostry","year":"2017","journal-title":"Mycotoxin Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1358","DOI":"10.1128\/jb.168.3.1358-1365.1986","article-title":"Osmotic Adjustment in the Filamentous Fungus Aspergillus-Nidulans","volume":"168","author":"Beever","year":"1986","journal-title":"J. Bacteriol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2179","DOI":"10.1093\/emboj\/16.9.2179","article-title":"The two isoenzymes for yeast NAD(+)-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation","volume":"16","author":"Ansell","year":"1997","journal-title":"Embo J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"878","DOI":"10.1111\/1462-2920.13617","article-title":"Glycerol metabolism and transport in yeast and fungi: Established knowledge and ambiguities","volume":"19","author":"Klein","year":"2017","journal-title":"Environ. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"5857","DOI":"10.1128\/AEM.05338-11","article-title":"Gpd1 and Gpd2 Fine-Tuning for Sustainable Reduction of Glycerol Formation in Saccharomyces cerevisiae","volume":"77","author":"Hubmann","year":"2011","journal-title":"Appl. Environ. Microb."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1515\/hsz-2014-0295","article-title":"Cytosolic thiol switches regulating basic cellular functions: GAPDH as an information hub?","volume":"396","author":"Hildebrandt","year":"2015","journal-title":"Biol. Chem."},{"key":"ref_13","first-page":"4135","article-title":"Gpd1, Which Encodes Glycerol-3-Phosphate Dehydrogenase, Is Essential for Growth under Osmotic-Stress in Saccharomyces cerevisiae, and Its Expression Is Regulated by the High-Osmolarity Glycerol Response Pathway","volume":"14","author":"Albertyn","year":"1994","journal-title":"Mol. Cell Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1099\/13500872-145-3-715","article-title":"Different signalling pathways contribute to the control of gene expression by osmotic stress in Saccharomyces cerevisiae","volume":"145","author":"Rep","year":"1999","journal-title":"Microbiology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1111\/j.1365-2958.1995.mmi_17010095.x","article-title":"Cloning and characterization of gpd2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in saccharomyces cerevisiae, and its comparison with gpd1","volume":"17","author":"Eriksson","year":"1995","journal-title":"Mol. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"39677","DOI":"10.1074\/jbc.M403310200","article-title":"Distinct intracellular localization of Gpd1p and Gpd2p, the two yeast isoforms of NAD-dependent glycerol-3-phosphate dehydrogenase, explains their different contributions to redox-driven glycerol production","volume":"279","author":"Valadi","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/0014-5793(91)80930-2","article-title":"Osmoregulation in Saccharomyces-Cerevisiae\u2014Studies on the Osmotic Induction of Glycerol Production and Glycerol 3-Phosphate Dehydrogenase (Nad+)","volume":"286","author":"Andre","year":"1991","journal-title":"FEBS Lett."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/s00203-024-04187-x","article-title":"Replacing Glycerol-3-Phosphate Dehydrogenase with NADH Oxidase: Effects on Glucose Fermentation and Product Formation in Saccharomyces cerevisiae","volume":"207","author":"Khattab","year":"2024","journal-title":"Arch. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1002\/(SICI)1097-0061(199707)13:9<783::AID-YEA128>3.0.CO;2-W","article-title":"Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase","volume":"13","author":"Michnick","year":"1997","journal-title":"Yeast"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1016\/j.funbio.2019.09.011","article-title":"Characterization of gfdB, putatively encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans","volume":"124","author":"Leiter","year":"2020","journal-title":"Fungal Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1046\/j.1365-2958.2001.02223.x","article-title":"Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus","volume":"39","author":"Fillinger","year":"2001","journal-title":"Mol. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2423","DOI":"10.1007\/s00253-023-12384-9","article-title":"Species-specific effects of the introduction of in osmophilic aspergilli","volume":"107","author":"Orosz","year":"2023","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2291","DOI":"10.1534\/g3.118.200253","article-title":"Predicted Glycerol 3-Phosphate Dehydrogenase Homologs and the Glycerol Kinase GIcA Coordinately Adapt to Various Carbon Sources and Osmotic Stress in Aspergillus fumigatus","volume":"8","author":"Zhang","year":"2018","journal-title":"G3-Genes Genomes Genet."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Hu, Y.L., Yang, G., Zhang, D.P., Liu, Y.J., Li, Y., Lin, G.L., Guo, Z.Q., Wang, S.H., and Zhuang, Z.H. (2018). The PHD Transcription Factor Rum1 Regulates Morphogenesis and Aflatoxin Biosynthesis in Aspergillus flavus. Toxins, 10.","DOI":"10.3390\/toxins10070301"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"404","DOI":"10.1006\/prep.2001.1588","article-title":"Osmoregulatory isoform of dihydroxyacetone phosphate reductase from Dunaliella tertiolecta: Purification and characterization","volume":"24","author":"Ghoshal","year":"2002","journal-title":"Protein Expres. Purif."},{"key":"ref_26","first-page":"112","article-title":"Histone Methyltransferase aflrmtA gene is involved in the morphogenesis, mycotoxin biosynthesis, and pathogenicity of Aspergillus flavus","volume":"134","author":"Zhuang","year":"2017","journal-title":"Toxicon"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Yang, K.L., Qin, Q.P., Liu, Y.H., Zhang, L.M., Liang, L.L., Lan, H.H., Chen, C.H., You, Y.C., Zhang, F., and Wang, S.H. (2016). Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus. Front. Cell. Infect. Microbiol., 6.","DOI":"10.3389\/fcimb.2016.00190"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Jia, K.Z., Yan, L.J., Jia, Y.P., Xu, S.T., Yan, Z.Q., and Wang, S.H. (2021). aflN Is Involved in the Biosynthesis of Aflatoxin and Conidiation in Aspergillus flavus. Toxins, 13.","DOI":"10.3390\/toxins13110831"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"7646","DOI":"10.1128\/AEM.02905-13","article-title":"Glycerol-3-phosphate Acyltransferase contributes to triacylglycerol biosynthesis, lipid droplet formation, and host invasion in Metarhizium robertsii","volume":"79","author":"Gao","year":"2013","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1111\/1751-7915.13701","article-title":"The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus","volume":"14","author":"Qin","year":"2021","journal-title":"Microb. Biotechnol."},{"key":"ref_31","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 method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1128\/MMBR.62.1.35-54.1998","article-title":"Asexual Sporulation in Aspergillus nidulans","volume":"62","author":"Adams","year":"1998","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1119","DOI":"10.1093\/gbe\/evaa107","article-title":"Recurrent Loss of, a Master Regulator of Asexual Development in Filamentous Fungi, Correlates with Changes in Genomic and Morphological Traits","volume":"12","author":"Mead","year":"2020","journal-title":"Genome Biol. Evol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1104","DOI":"10.1128\/EC.00069-12","article-title":"NsdC and NsdD Affect Morphogenesis and Aflatoxin Production","volume":"11","author":"Cary","year":"2012","journal-title":"Eukaryot. Cell"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"103478","DOI":"10.1016\/j.fgb.2020.103478","article-title":"Genetic map and heritability of Aspergillus flavus","volume":"144","author":"Gell","year":"2020","journal-title":"Fungal Genet. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Sweany, R.R., and Damann, K.E. (2020). Influence of Neighboring Clonal-Colonies on Aflatoxin Production by Aspergillus flavus. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.03038"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.1046\/j.1365-2958.2000.01955.x","article-title":"The control of intracellular glycerol in influences osmotic stress response and resistance to increased temperature","volume":"36","author":"Siderius","year":"2000","journal-title":"Mol. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1111\/j.1432-1033.1980.tb04798.x","article-title":"Prediction of Secondary Structural Elements in Glycerol-3-Phosphate Dehydrogenase by Comparison with Other Dehydrogenases","volume":"109","author":"Otto","year":"1980","journal-title":"Eur. J. Biochem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1002\/jat.3422","article-title":"Aflatoxin biomarkers in hair may facilitate long-term exposure studies","volume":"37","author":"Mupunga","year":"2017","journal-title":"J. Appl. Toxicol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Shi, Y.K., Wang, H., Yan, Y.X., Cao, H.J., Liu, X.H., Lin, F.C., and Lu, J.P. (2018). Glycerol-3-Phosphate Shuttle Is Involved in Development and Virulence in the Rice Blast Fungus. Front. Plant Sci., 9.","DOI":"10.3389\/fpls.2018.00687"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1616","DOI":"10.1128\/EC.00193-09","article-title":"Our paths might cross: The role of the fungal cell wall integrity pathway in stress response and cross talk with other stress response pathways","volume":"8","author":"Fuchs","year":"2009","journal-title":"Eukaryot. Cell"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1111\/mmi.13484","article-title":"The Aspergillus fumigatus SchA(SCH9) kinase modulates SakA(HOG1) MAP kinase activity and it is essential for virulence","volume":"102","author":"Dolan","year":"2016","journal-title":"Mol. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1016\/j.funbio.2019.10.002","article-title":"Crosstalk between Saccharomycescerevisiae SAPKs Hog1 and Mpk1 is mediated by glycerol accumulation","volume":"124","author":"Laz","year":"2020","journal-title":"Fungal Biol."}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/8\/610\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T10:59:24Z","timestamp":1786705164000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/8\/610"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,14]]},"references-count":43,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["jof12080610"],"URL":"https:\/\/doi.org\/10.3390\/jof12080610","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,14]]}}}