Adaptation of the Agrobacterium-mediated transformation method to the ligD-deficient Aspergillus oryzae NsPlD1 strain
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Abstract
Aspergillus oryzae is widely used to produce traditional fermented foods in Asian countries. It is also exploited as a host for homologous and heterologous protein production. The ligD-deficient A. oryzae NsPlD1 strain is a uridine/uracil auxotrophic and is commonly employed for gene targeting and recombinant expression. However, studies on the NsPlD1 strain were mainly supported by protoplast-mediated transformation. In the present study, we adapted the Agrobacterium tumefaciens-mediated transformation (ATMT) method using two auxotrophic selection markers to the A. oryzae strain. Notably, a histidine auxotrophic mutant was constructed by deleting the hisB gene in the NsPlD1 strain. Under the appropriate conditions, the ATMT yields of the auxotrophic NsPlD1 strain reached 130 ± 10 transformants per 107 spores with the pyrG marker and 73 ± 18 transformants per 106 spores with the hisB marker. Furthermore, we successfully expressed the red fluorescent protein (DsRed) from Discosoma coral in the NsPlD1 strain with the ATMT method. Our work provided an additional option for genetic manipulation in the A. oryzae strain based on the pyrG and hisB auxotrophic markers.
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