Production of M-/GM-group aflatoxins catalyzed by the OrdA enzyme in aflatoxin biosynthesis

Abstract

Aspergillus parasiticus produces the minor aflatoxins M1 (AFM1), M2 (AFM2), GM1 (AFGM1), and GM2 (AFGM2), as well as the major aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2). Feeding of A. parasiticus with aspertoxin (12c-hydroxyOMST) caused AFM1 and AFGM1, and cell-free experiments using the microsomal fraction of A. parasiticus and aspertoxin caused production of AFM1, indicating that aspertoxin is a precursor of AFM1 and AFGM1. Feeding of the same fungus with O-methylsterigmatocystin (OMST) caused AFM1 and AFGM1 together with AFB1 and AFG1; feeding with dihydroOMST (DHOMST) caused AFM2 and AFGM2 together with AFB2 and AFG2. Incubation of either the microsomal fraction or OrdA enzyme-expressing yeast with OMST caused production of aspertoxin together with AFM1 and AFB1. These results demonstrated that the OrdA enzyme catalyzes both 12c-hydroxylation reaction from OMST to aspertoxin and the successive reaction from aspertoxin to AFM1. In contrast, feeding of the fungus with AFB1 did not produce any AFM1, demonstrating that M-/GM-aflatoxins are not produced from B-/G-aflatoxins. Furthermore, AFM1 together with AFB1 and AFG1 was also produced from 11-hydroxyOMST (HOMST) in feeding experiment of A. parasiticus, whereas no aflatoxins were produced when used the ordA deletion mutant. These results demonstrated that OrdA enzyme can also catalyze 12c-hydroxylation of HOMST to produce 11-hydroxyaspertoxin, which serves as a precursor for the production of AFM1 and AFGM1. The same pathway may work for the production of AFM2 and AFGM2 from DHOMST and dihydroHOMST through the formation of dihydroaspertoxin and dihydro-11-hydroxyaspertoxin, respectively.