Submitted to: Journal of Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/20/2003
Publication Date: 1/5/2004
Citation: Chang, P.-K. 2004. Lack of interaction between AFLR and AFLJ contributes to nonaflatoxigenicity of Aspergillus sojae. Journal of Biotechnology. 107:245-253. Interpretive Summary: Soy sauce consumption in the U.S. has been increasing steadily since 1970, with a market growth rate of 5 to 8 % annually. Currently, soy sauce has a market value of about 900 million dollars in the U.S. and about 5 billion dollars worldwide. Aspergillus sojae is a koji mold mainly used in commercial soy sauce fermentation. This mold is a close relative of the aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus, but has lost its ability to make the cancer causing compounds aflatoxins. However, two fundamental questions remain: what made A. sojae lose its ability to make the toxins and what is the probability for it to regain its aflatoxin-producing ability? In this study, the underlying mechanism responsible for the lack of aflatoxin production in A. sojae was elucidated. It was found that A. sojae had a defective AFLR regulatory protein, whose intact form is critical for making contact with another protein, AFLJ, which is a co-regulator with AFLR in initializing aflatoxin biosynthesis. The successful elucidation of the molecular mechanisms responsible for the nonaflatoxigenicty of A. sojae not only assures its safe use in food fermentation, but also has removed perceived public health concerns.
Technical Abstract: 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 regarded 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 aflR 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 aflJ differs from A. parasiticus aflJ 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 aflR, aflJ, pksA, nor1, ver1 and omtA in A. sojae as determined by real-time reverse transcriptase-polymerase chain reaction 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.