Norsolorinic Acid Mutant of Aspergillus flavus

Abstract

A mutant of Aspergillus Javus accumulating norsolorinic acid and approximately 50 % less aflatoxin than the parent strain was recovered after treatment of conidia with Nmethyl-N'-nitro-N-nitrosoguanidine. The gene nor controlling this mutant phenotype was recessive and linked to aJl-1 and leu on linkage group VII. Diploids homozygous for nor were similar to haploids in norsolorinic acid accumulation. The analysis of recombinant diploids and haploids showed that aJ-1 and nor were both distal to leu on the same chromosome arm. I N T R O D U C T I O NAflatoxins, secondary metabolites produced by Aspergillus Javus and Aspergillus parasiticus, have attracted considerable attention, and much has been reported about their biosynthesis and biological effects. Several intermediates in the aflatoxin biosynthetic pathway have been established through studies of induced mutants (Hsieh et al., 1976). Lee et al. (197 1 ) recovered a mutant of A . parasiticus which accumulated norsolorinic acid and less aflatoxin than the wild-type. This mutant was recessive in diploids (Bennett, 1979). Several aflatoxin mutants having no detectable pigmented intermediates have been recovered in A.Javus (Papa, 1979). This paper describes the genetic analysis of a norsolorinic acid mutant of A . Jlavus, including linkage detection and mapping. M E T H O D SStrains. Five auxotrophic strains of A.Javus having either white (w) or tan ( t ) spore colour were used in this study. Their individual genotypes were as follows: 1. w lys (requiring lysine); 2. tpdx leu (pyridoxine, leucine); 3. I leu; 4. afl-1 t leu; and 5 . t arg (arginine). Strain 4 was also impaired in aflatoxin production (aJ-1 ). The induction of auxotrophy, spore colour variation, and aflatoxin mutants has been described previously (Leaich & Papa, 1974; Papa, 1976 Papa, , 1979.Following the exposure of conidia from strain 1 to N-methyl-N'-nitro-N-nitrosoguanidine (NG), a mutant, designated l-N and having an orange-red mycelium, was recovered. The gene controlling the production of this orange-red pigment (norsolorinic acid) has been designated nor (Bennett, 1979).Media. The complete medium (CM) consisted of Czapek-Dox broth, 0.75 % (w/v) malt extract, 0.25 % (w/v) yeast extract and 1.5% (w/v) agar. The minimal medium (MM) was CM without the malt and yeast extracts. Diploids were plated on CM containing 0.007 % (w/v) p-fluorophenylalanine to induce haploidization. Appropriately supplemented MM was used for the identification of nutritional requirements of haploid segregants. The medium for production of aflatoxin consisted of 2 % (w/v) yeast extract and 20% (w/v) sucrose.Aflatoxin and norsolorinic acid determinations. Aflatoxin and norsolorinic acid were extracted according to procedures described by Lillard et al. (1970) and Papa (1976). Extracts were suspended in chloroform, appropriately diluted, and spotted on thin-layer chromatographic plates (pre-coated silica gel) from EM Laboratories, Elmsford, N.Y., U.S.A. Chromatograms for aflatoxin and norsolorinic acid separations were...

Isolation and Characterization of the Versicolorin B Synthase Gene from Aspergillus parasiticus

“…In contrast to the earlier findings of Papa (53)(54)(55), whose work identified several linkage groups in A. parasiticus for AFB 1 -related genes, it appears that like a growing number of other secondary metabolites, aflatoxin is a further example of a natural product whose biosynthetic genes are clustered. These workers have identified the organization of several demonstrated and pre- (Fig.…”

“…Preliminary evidence has been gathered to suggest that these genes are substantially clustered (48 -51), contrary to earlier reports (52)(53)(54)(55). A probable polyketide synthase (pksA) and two fatty acid synthase (fas-1A and fas-2A) genes have been identified by sequence homology and gene disruption experiments (51).…”

Sclerotia Formation and Toxin Production in Large Sclerotial <i>Aspergillus flavus</i> Isolates from Kenya

“…Aspergillus flavus isolates were analyzed for the presence of two aflatoxin biosynthesis genes in relation to their capability to produce aflatoxins, targeting the structural genes aflD, and aflQ genes. The aflD gene encodes an enzyme that catalyzes the conversion of the first stable aflatoxin biosynthesis intermediate, norsolorinic acid to averantin [5] while the aflQ gene is involved in the conversion of O-methylsterigmatocystin (omst) to aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) and dihydro-O-methylsterigmatocystin (dmdhst) to aflatoxin B2 (AFB2) and aflatoxin G2 (AFG2) [6].…”

Silencing of the Aflatoxin Gene Cluster in a Diploid Strain of Aspergillus flavus Is Suppressed by Ectopic aflR Expression

“…Through this cycle, mutations can be assessed for dominance and loci can be mapped as linkage groups. Papa performed parasexual crosses between wild-type PC-7 and mutant strains derived from PC-7, and was able to assign 36 loci to eight linkage groups (Papa 1973(Papa , 1979(Papa , 1980(Papa , 1982(Papa , 1986. The loci included genes for spore color, nutrient utilization, and aflatoxin biosynthesis.…”