Function Analysis Of Biotin Biosynthetic Gene BioDA In Aspergillus Flavus

Aspergillus a widely existing soil saprophytic fungus which causes diseases in human and animals.Aflatoxins produced by Aspergillus flavus are considered one of the strongest carcinogens in the world.Biotin,an essential enzyme cofactor,is required for basic life activities such as carboxylation and decarboxylation.The complete system of biotin biosysthesis is existed in fungi and bacterial except mammals.In this paper,AfbioDA which plays a key role in biontin biosysthesis was studied for the first time in Aspergillus flavus.The AFLA₀49090 gene found by bioinformatic analysis in Aspergillus flavus data base encodes AfBioDA sharing high conservation with EcBioA and EcBioD at animo acid sequence and structure.Expression of AfbioDA allows growth of E.coli ?EcbioA and ?EcbioD strains on biotin-free medium indicating function equivalence of AfBioDA to Diaminopelargonic Acid Synthase and dethiobiotin synthetase.Gel filtration chromatography and chemical crosslinking test revealed that AfBioDA could exist as monomer,dimer and even polymer in vivo.The subcellular localization test proved that AfBioDA transferred into mitochondria using N-terminal signal peptide.The gene AfbioDA knockout strain(?AfbioDA)and complementation strain(C?AfbioDA)were constructed to analyze whether or not this gene affects growth and virul-ence in Aspergillus flavus.The ?AfbioDA strain could't grow on biotin-free medium explaining that the gene AfbioDA involves in the biotin biosynthesis on biotin-free medium.The phenotypic experiments showed that AfbioDA gene reduced slightly production of conidia,synthesis of aflatoxin and infection of peanuts except growth in rich medium.In summary,AfBioDA catalyzed the synthesis of biotin precursors 7-8-diaminonanoic acid and dethiobiotin.AfBioDA protein can exist as monomer,dimer and polymer in the mitochondria.The AfbioDA gene deletion decreased the virulence of Aspergillus flavus.These findings significantly update our former knowledge to fungal biotin biosysthesis and provide theoretical basis for designing inhibitory drug to eliminate Aspergillus flavus infections in the future.