Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 24, 2004
Publication Date: November 1, 2004
Citation: Ehrlich, K.C., Chang, P.-K., Yu, J., Cotty, P.J. 2004. Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation. Applied and Environmental Microbiology. 70(11):6518-6524. Interpretive Summary: Aspergillus flavus is a mold that can contaminate corn and cottonseed. It produces aflatoxin B1, which is a highly toxic and carcinogenic chemical. Contamination of foods with aflatoxin B1 makes them unsafe for animal and human consumption. Other molds make aflatoxins B1 and G1. Aflatoxin G1 is even more toxic and carcinogenic than aflatoxin B1. Cotton farmers would benefit from development of methods to reduce aflatoxin contamination since contamination is an endemic problem in cotton in Arizona and Texas. We have now found the reason why Aspergillus flavus makes only aflatoxin B1. It is missing the critical piece of DNA (gene) that converts a precursor to aflatoxin G1. We found this result first by comparing the sequence of the large piece of DNA that contains all of the genes needed to make both types of aflatoxin to the DNA sequence of A.flavus which only makes aflatoxin B1. We found that A. flavus is missing portions of two genes (cypA and norB) which make two different types of oxidative enzymes. When the gene cypA was disrupted, the fungi with the disrupted gene could not make aflatoxin G1, but still made B1. This result proves that cypA is needed for aflatoxin G1 production. Understanding how Aspergillus flavus is different from other Aspergillus species will aid in understanding the populations of fungi that contribute to contamination of cottonseed and will help in developing strategies to reduce contamination in Arizona and Texas cottonseed.
Technical Abstract: Aspergillus flavus isolates produce only B-type aflatoxins while A. parasiticus and A. nomius produce both B- and G-type aflatoxins. Sequence comparison of the aflatoxin pathway gene cluster from these species revealed that A. flavus isolates are missing portions of two putative biosynthesis genes located in the cluster upstream from the polyketide synthase gene, pksA. One of the missing genes (cypA) is predicted to encode a cytochrome P450 monooxygenase unique to the cluster while the other gene, norB, is predicted to encode an aryl alcohol dehydrogenase which is highly homologous to the aflatoxin cluster gene, norA. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G-type, but not B-type aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Tri4 (34%), a Fusarium P450 monooxygenase involved in the trichodiene epoxidation. The substrate for CypA may be a 1-methoxy-1-buten-4-carboxylic acid intermediate formed after oxidative cleavage of the A-ring of O-methylsterigmatocystin (OMST) by another P450 monooxygenase, OrdA, which is required for formation of aflatoxins B1 and B2. We propose that formation of G-type aflatoxins requires the CypA-catalyzed epoxidation of the double bond in this intermediate.