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 (ΔgfdA, ΔgfdB), double-gene knockout strains (ΔgfdAΔgfdB) and their corresponding complemented strains (gfdA^C, gfdB^C) 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 ΔgfdAΔgfdB strains is almost similar to that of the ΔgfdA 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 ΔgfdA and ΔgfdAΔgfdB 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.

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