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Characterization of the Bifunctional Enzyme BioDA Involved in Biotin Synthesis and Pathogenicity in Aspergillus flavus.

Biotin is an important enzyme cofactor that plays a key role in all three domains. The classical bifunctional enzyme BioDA in eukaryotes (such as Aspergillus flavus and Arabidopsis thaliana) is involved in the antepenultimate and penultimate steps of biotin biosynthesis. In this study, we identified a A. flavus bifunctional gene bioDA which could complement both Escherichia coli ΔEcbioD and ΔEcbioA mutants. Interestingly, the separated domain of AfBioD and AfBioA could, respectively, fuse with EcBioA and EcBioD well and work together. What is more, we found that BioDA was almost localized to the mitochondria in A. flavus, as shown by N-terminal red fluorescent protein tag fusion. Noteworthy, the subcellular localization of AfBioDA is never affected by common environmental stresses (such as hyperosmotic stress or oxidative stress). The knockout strategy demonstrated that the deletion of AfbioDA gene from the chromosome impaired the biotin de novo synthesis pathway in A. flavus. Importantly, this A. flavus mutant blocked biotin production and decreased its pathogenicity to infect peanuts. Based on the structural comparison, we found that two inhibitors (amiclenomycin and gemcitabine) could be candidates for antifungal drugs. Taken together, our findings identified the bifunctional AfbioDA gene and shed light on biotin biosynthesis in A. flavus.