Demonstration of a highly efficient fungal transformation method used to target four genes involved in conidial pigment biosynthesis

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The development of a highly efficient gene‐targeting approach is a prerequisite to the success of functional genomics of Aspergillus flavus, an aflatoxin‐producing fungus of great economic importance. To this end, the ku70 gene, a gene of the nonhomologous end‐joining pathway was deleted to increase the homologous recombination frequency. The pyrG gene in the resulting strain was disrupted by a gene knock‐in strategy for using the A. parasiticus pyrG gene as a selectable marker for transformation. The gene‐targeting frequencies of the A. flavus br1, br2, wA and ygA genes, homologous to genes reported to be involved in conidial pigment biosynthesis in A. fumigatus or A. nidulans, were examined in the A. flavus ?ku70?pyrG strain. The br1 gene is located about 100 kb from the clustered br2 and wA genes on chromosome IV, and the ygA gene is located on chromosome I. The gene‐targeting frequencies were achieved at 80 to 100% as confirmed by PCR screenings of altered genomic patterns of the transformants. Disruption of br2 and wA resulted in mutants of white and olive‐green conidial color, respectively. The br1 and ygA mutants retained the parental strain's yellowish green conidial color. This system substantially reduces the time and workload necessary to obtain knockout mutants. It will expedite the progresses in the functional genomic studies of A. flavus.