Traditional molecular techniques have been used in research in discovering the genes and enzymes that are involved in aflatoxin formation and genetic regulation. We cloned most, if not all, of the aflatoxin pathway genes. A consensus gene cluster for aflatoxin biosynthesis was discovered in 2005. The factors that affect aflatoxin formation have been studied. In this report, the author summarized the current status of research progress and future possibilities that may be used for solving aflatoxin contamination.

https://www.mdpi.com/2072-6651/4/11/1024/pdf

Current Understanding on Aflatoxin Biosynthesis and Future Perspective in Reducing Aflatoxin Contamination

DNA isolated from the wild-type aflatoxin-producing (Afl+) fungus Aspergillus parasiticus NRRL 5862 was used to construct a cosmid genomic DNA library employing the homologous gene (pyrG) encoding orotidine monophosphate decarboxylase for selection of fungal transformants. The cosmid library was transformed into an Afl- mutant, A. parasiticus CS10 (ver-1 wh-1 pyrG), deficient in the conversion of the aflatoxin biosynthetic intermediate versicolorin A to sterigmatocystin. One pyrG+ Afl+ transformant was identified. DNA fragments from this transformant, recovered by marker rescue, contained part of the cosmid vector including the pyrG gene, the ampr gene, and a piece of the original genomic insert DNA. Transformation of these rescued DNA fragments into A. parasiticus CS10 resulted in production of wild-type levels of aflatoxin and abundant formation of sclerotia. The gene responsible for this complementation (ver-1) was identified by Northern RNA analysis and transformation with subcloned DNA fragments. The approximate locations of transcription initiation and polyadenylation sites of ver-1 were determined by an RNase protection assay and cDNA sequence analysis. The predicted amino acid sequence, deduced from the ver-1 genomic and cDNA nucleotide sequences, was compared with the EMBL and GenBank data bases. The search revealed striking similarity with Streptomyces ketoreductases involved in polyketide biosynthesis. Images

Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis.

Aflatoxins in stored maize and rice grains in Liaoning Province, China

Two genes involved in aflatoxin B1 (AFB1) biosynthesis in Aspergillus parasiticus, nor-1 and ver-1, were localized to a 35-kb region on one A. parasiticus chromosome and to the genomic DNA fragment carried on a single cosmid, NorA. A physical and transcriptional map of the 35-kb genomic DNA insert in cosmid NorA was prepared to help determine whether other genes located in the nor-1-ver-1 region were involved in aflatoxin synthesis. Northern (RNA) analysis performed on RNA isolated from A. parasiticus SU1 grown in aflatoxin-inducing medium localized 14 RNA transcripts encoded by this region. Eight of these transcripts, previously unidentified, showed a pattern of accumulation similar to that of nor-1 and ver-1, suggesting possible involvement in AFB1 synthesis. To directly test this hypothesis, gene-1, encoding one of the eight transcripts, was disrupted in A. parasiticus CS10, which accumulates the aflatoxin precursor versicolorin A, by insertion of plasmid pAPNVES4. Thin-layer chromatography revealed that gene-1 disruptant clones no longer accumulated versicolorin A. Southern hybridization analysis of these clones indicated that gene-1 had been disrupted by insertion of the disruption vector. These data confirmed that gene-1 is directly involved in AFB1 synthesis. The predicted amino acid sequence of two regions of gene-1 showed a high degree of identity and similarity with the beta-ketoacyl-synthase and acyltransferase functional domains of polyketide synthases, consistent with a proposed role for gene-1 in polyketide backbone synthesis.

Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in the aflatoxin pathway.

The role of zinc on the primary metabolism of Aspergillus parasiticus in relation to aflatoxin biosynthesis was studied. Zinc deficiency impaired growth and metabolism of nucleic acid and protein while stimulating that of lipid. The activities of enzymes of glycolytic cycle were affected indicating that these are zinc dependent enzymes. Impaired glycolytic cycle tends to reduce the level of primary metabolites like pyruvate, citrate and oxaloacetate which trigger aflatoxin formation. During stationary phase these precursors accumulate as these are not used up for the synthesis of secondary metabolites (aflatoxins) due to zinc deficiency. ATP and energy-charge (E.C.) which are important for various physiological processes like growth are reduced in zinc deficiency. Also zinc deficiency results in low levels of AMP which favour synthesis of lipid. Inorganic phosphate accumulated due to zinc deficiency and this may be unfavourable for aflatoxin synthesis.

https://mmbr.asm.org/content/mmbr/41/4/822.full.pdf

Biosynthesis of aflatoxins.

A mutant formed after UV irradiation of a potent aflatoxin producing strain ofAspergillus parasiticus elaborated 80% less aflatoxin than did the parent strain and produced an orange-red pigment. This new metabolite which represents 1% of the mycelial mass has been identified as 2-hexanoyl-1,3,6,8-tetrahydroxyanthraquinone (norsolorinic acid), mol wt 370, mp 256–257 C, and molecular formula C20H18O7.

Norsolorinic acid from a mutant strain ofAspergillus parasiticus

Mutations affecting the production of aflatoxin might be useful in studying its biosynthesis, and in understanding the wide variation in aflatoxigenicity of fungal strains isolated from nature. Two methods of isolating mutants of Aspergillus in which aflatoxin production is attenuated or lost are presented. In one method mutants are selected on the basis of altered fluorescence under ultraviolet light. The second method utilizes a mutant strain which produces aflatoxin and an orange-red mycelial pigment not detected in the wild type. Unpigmented mutants derived from this strain exhibit lowered or lost aflatoxigenicity.

L. A. Goldblatt

Papers

Norsolorinic acid from a mutant strain ofAspergillus parasiticus

The isolation of mutants of Aspergillus flavus and A.parasiticus with altered aflatoxin producing ability.