Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species.
The biosynthesis of aflatoxins (B(1), G(1), B(2), and G(2)) is a multi-enzyme process controlled genetically by over 20 genes. In this study, we report the identification and characterization of the avfA gene, which was found to be involved in the conversion of averufin (AVF) to versiconal hemiacetal acetate (VHA), in Aspergillus parasiticus and A. flavus; a copy of avfA gene was also cloned from a non-aflatoxin producing strain A. sojae. Complementation of an averufin-accumulating, non-aflatoxigenic mutant strain of A. parasiticus, SRRC 165, with the avfA gene cloned from A. flavus, restored the ability of the mutant to convert AVF to VHA and to produce aflatoxins B(1), G(1), B(2), and G(2). Sequence analysis revealed that a single amino acid replacement from aspartic acid to asparagine disabled the function of the enzyme in the mutant strain SRRC 165. The A. parasiticus avfA was identified to be a homolog of previously sequenced, but functionally unassigned transcript, stcO, in A. nidulans based on sequence homology at both nucleotide (57%) and amino acid (55%) levels. In addition to avfA, another aflatoxin pathway gene, omtB, encoding for an O-methyltransferase involved in the conversion of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST), was cloned from A. parasiticus, A. flavus, and A. sojae. The omtB gene was found to be highly homologous to stcP from A. nidulans, which has been reported earlier to be involved in a similar enzymatic step for the sterigmatocystin formation in that species. RT-PCR data demonstrated that both the avfA and avfA1 as well as omtB genes in A. parasiticus were expressed only in the aflatoxin-conducive medium. An analysis of the degrees of homology for the two reported genes between the Aspergillus species A. parasiticus, A. flavus, A. nidulans and A. sojae was conducted.
Yu
J
J
Southern Regional Research Center, USDA, Agricultural Research Service, New Orleans, LA 70179, USA. jiuyu@commserver.srrc.usda.gov
Woloshuk
C P
CP
Bhatnagar
D
D
Cleveland
T E
TE
D000348:Aflatoxins / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D000348:Aflatoxins; D000880:Anthraquinones; D004271:DNA, Fungal; D005656:Fungal Proteins; C027121:versiconal hemiacetal acetate; C029856:averufin; D010088:Oxidoreductases; C410102:avfA protein, Aspergillus parasiticus; D008780:Methyltransferases; C410091:omtB protein, Aspergillus