Lack of interaction between AFLR and AFLJ contributes to nonaflatoxigenicity of Aspergillus sojae

Aspergillus sojae, which is believed to be a domesticated strain of Aspergillus parasiticus, contains all of the aflatoxin biosynthetic genes but is unable to produce aflatoxins and is generally recognized as safe (GRAS) for producing fermented foods. In A. parasiticus both aflR, the aflatoxin pathway-specific regulatory gene, and aflJ, a co-activator gene, are necessary for transcription of genes encoding the aflatoxin biosynthetic enzymes. A. sojae aflR differs from A. parasiticus afR in that it encodes extra His and Ala, and has a pretermination defect that causes truncation of the carboxyl terminus of the predicted protein. A. sojae afJ differs from A. parasiticus afJ in that it encodes a predicted protein with Ser39 replaced by Ala and Ser283 replaced by Pro. Steady-state levels of aflatoxin biosynthetic gene transcripts of afR, afJ, pksA, norl, verl and omtA in A. sojae as determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were much lower than those of A. parasiticus. Yeast two-hybrid assays showed that the truncated A. sojae AFLR did not interact with AFLJ of A. sojae and A. parasiticus but that an A. sojae AFLR reverted to the putative ancestral form interacted normally with AFLJ of A. sojae and A. parasiticus. Deletion analysis showed that both amino- and carboxy-terminal regions of the A. sojae AFLJ were important for the R-J interaction. The truncated A. sojae AFLR thus not only was impaired in its ability to activate transcription of aflatoxin biosynthetic genes, but also was unable to interact with AFLJ, in A. parasiticus both of which are required for normal expression of the aflatoxin biosynthetic genes. Consequently, the lack of aflatoxin-producing ability of A. sojae resulted primarily from two defects in the regulatory mechanism responsible for gene transcription. Published by Elsevier B.V.