Aspergillus flavus mutant strain 649, which has a genomic DNA deletion of at least 120 kb covering the aflatoxin biosynthesis cluster, was transformed with a series of overlapping cosmids that contained DNA harboring the cluster of genes. The mutant phenotype of strain 649 was rescued by transformation with a combination of cosmid clones 5E6, 8B9, and 13B9, indicating that the cluster of genes involved in aflatoxin biosynthesis resides in the 90 kb of A. flavus genomic DNA carried by these clones. Transformants 5E6 and 20B11 and transformants 5E6 and 8B9 accumulated intermediate metabolites of the aflatoxin pathway, which were identified as averufanin and/or averufin, respectively. These data suggest that avf1, which is involved in the conversion of averufin to versiconal hemiacetal acetate, was present in the cosmid 13B9. Deletion analysis of 13B9 located the gene on a 7-kb DNA fragment of the cosmid. Transformants containing cosmid 8B9 converted exogenously supplied O-methylsterigmatocystin to aflatoxin, indicating that the oxidoreductase gene (ord1), which mediates the conversion of O-methylsterigmatocystin to aflatoxin, is carried by this cosmid. The analysis of transformants containing deletions of 8B9 led to the localization of ord1 on a 3.3-kb A. flavus genomic DNA fragment of the cosmid.

Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Most of the genes involved in the biosynthesis of aflatoxin are contained within a single cluster in the genome of these filamentous fungi. Studies directed toward understanding the molecular biology of aflatoxin biosynthesis have led to a number of important discoveries. A pair of fatty acid synthase genes were identified that are involved uniquely in aflatoxin biosynthesis. Two genes were also characterized that represent new families of cytochrome P450 monooxygenases. Gene expression is coordinated during aflatoxin production and is under the control of a positive regulatory gene belonging to a family of fungal transcriptional activators associated with various metabolic pathways in fungi.

Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O-methylsterigmatocystin to aflatoxin has been proposed to be catalyzed by an oxidoreductase. Transformants of Aspergillus flavus 649WAF2 containing a 3.3-kb genomic DNA fragment and the aflatoxin biosynthesis regulatory gene aflR converted exogenously supplied O-methylsterigmatocystin to aflatoxin B 1 . A gene, ord1, corresponding to a transcript of about 2 kb was identified within the 3.3-kb DNA fragment. The promoter region presented a putative AFLR binding site and a TATA sequence. The nucleotide sequence of the gene revealed an open reading frame encoding a protein of 528 amino acids with a deduced molecular mass of 60.2 kDa. The gene contained six introns and seven exons. Heterologous expression of the ord1 open reading frame under the transcriptional control of the Saccharomyces cerevisiae galactose-inducible gal1 promoter results in the ability to convert O-methylsterigmatocystin to aflatoxin B 1 . The data indicate that ord1 is sufficient to accomplish the last step of the aflatoxin biosynthetic pathway. A search of various databases for similarity indicated that ord1 encodes a cytochrome P-450-type monooxygenase, and the gene has been assigned to a new P-450 gene family named CYP64.

Summary The effect of constitutive expression of nitrate reductase (NR) on the regulation pattern of nitrite and nitrate transporters (NT) has been analysed in Chlamydomonas reinhardtii. Strain Tx11‐8, bearing the chimeric Nia1 gene for NR under the control of the cabll‐1 gene promoter, showed a constitutive NR which was inactive in ammonium‐grown cells. In contrast to the wild‐type, incubation of Tx11‐8 in ammonium media containing nitrate at micromolar concentrations resulted in reactivation of inactive NR and excretion of nitrite. In addition, negligible amounts of NT gene transcripts were induced by nitrate in this strain. However, NT transcripts from Tx11‐8 cells were induced in amounts similar to those in the wild‐type, by preventing constitutive NR expression from the cabll‐1 gene promoter in the dark. Strains lacking high‐affinity NT and nitrite reductase activity and having the chimeric Nia1 gene have been constructed by genetic crosses. All these strains were sensitive to chlorate in ammonium‐containing media and excreted nitrite when nitrate was present at millimolar concentrations. Spontaneous chlorate‐resistant mutants isolated from these strains lacked constitutive NR activity though they constitutively expressed NR mRNA. The data strongly suggest that nitrate plays an important post‐transcriptional regulatory role and that NR is involved in the regulation of the nitrate/ nitrite transport activities in C. reinhardtii.

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