Development of a homologous transformation system for Aspergillus parasiticus with the gene encoding nitrate reductase

Horng, J S; Chang, Perng‐Kuang; Pestka, James J.; Linz, John E.

doi:10.1007/bf00271564

Cited by 57 publications

(40 citation statements)

References 18 publications

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“…2A). Transformation of A. parasiticus CS10-N2 was carried out by a protoplast-polyethylene glycol method as previously described (10). Czapek Solution (CZ) agar (Difco) supplemented with Cove's trace-element solution (7), 20 mM uracil, and 0.6 M KCl was used for the regeneration of protoplasts after transformation.…”

Section: Methodsmentioning

confidence: 99%

Nonfunctionality of Aspergillus sojae aflR in a Strain of Aspergillus parasiticus with a Disrupted aflR Gene

Takahashi

Chang

Matsushima

et al. 2002

Appl Environ Microbiol

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Aspergillus sojae belongs to the Aspergillus section Flavi but does not produce aflatoxins. The functionality of the A. sojae aflR gene (aflRs) was examined by transforming it into an ⌬aflR strain of A. parasiticus, derived from a nitrate-nonutilizing, versicolorin A (VERA)-accumulating strain. The A. parasiticus aflR gene (aflRp) transformants produced VERA, but the aflRs transformants did not. Even when aflRs was placed under the control of the amylase gene (amyB) promoter of Aspergillus oryzae, the amy(p)::aflRs transformants did not produce VERA. A chimeric construct containing the aflRs promoter plus the aflRs N-and aflRp C-terminal coding regions could restore VERA production, but a construct containing the aflRp promoter plus the aflRp N-and aflRs C-terminal coding regions could not. These results show that the A. sojae aflR promoter is functional in A. parasiticus and that the HAHA motif does not affect the function of the resulting hybrid AflR. We conclude that the lack of aflatoxin production by A. sojae can be attributed, at least partially, to the premature termination defect in aflRs, which deletes the C-terminal transcription activation domain that is critical for the expression of aflatoxin biosynthetic genes.

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Section: Methodsmentioning

confidence: 99%

Nonfunctionality of Aspergillus sojae aflR in a Strain of Aspergillus parasiticus with a Disrupted aflR Gene

Takahashi

Chang

Matsushima

et al. 2002

Appl Environ Microbiol

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“…Two strains of A. parasiticus were used for this research: NRRL2999, a wild-type strain, well documented for AF production (1, 2); and strain B62 (brn; nor-1; niaD) (33). The brn mutation in B62 results in brown conidia.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus

1995

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Aflatoxins are potent toxic and carcinogenic compounds, produced by Aspergillus parasiticus and A. flavus as secondary metabolites. In this research, a polyketide synthase gene (pksL1), the key gene for aflatoxin biosynthesis initiation in A. parasiticus, has been functionally identified and molecularly characterized. PCRderived DNA probes were used to find the pksL1 gene from subtracted, aflatoxin-related clones. Gene knockout experiments generated four pksL1 disruptants which lost both the ability to produce aflatoxins B1, B2, and G1 and the ability to accumulate norsolorinic acid and all other intermediates of the aflatoxin biosynthetic pathway. A pksL1 DNA probe detected a 6.6-kb poly(A) ؉ RNA transcript in Northern (RNA) hybridizations. This transcript, associated with aflatoxin production, exhibited a regulated expression that was influenced by growth phase, medium composition, and culture temperature. DNA sequencing of pksL1 revealed an open reading frame for a polypeptide (PKSL1) of 2,109 amino acids. Sequence analysis further recognized four functional domains in PKSL1, acyl carrier protein, ␤-ketoacyl-acyl carrier protein synthase, acyltransferase, and thioesterase, all of which are usually present in polyketide synthases and fatty acid synthases. On the basis of these results, we propose that pksL1 encodes the polyketide synthase which synthesizes the backbone polyketide and initiates aflatoxin biosynthesis. In addition, the transcript of pksL1 exhibited heterogeneity at the polyadenylation site similar to that of plant genes.Aflatoxins (AFs) are a group of polyketide-derived mycotoxins produced by certain strains of Aspergillus parasiticus and A. flavus (10, 27). As with other secondary metabolites, the biosynthesis of AFs occurs at the beginning of and during idiophase when growth of the molds has greatly slowed or stopped, while developmental and chemical differentiation ensue (27). AFB 1 , AFB 2 , AFG 1 , and AFG 2 are the most abundant AFs (20). These toxic and carcinogenic compounds frequently contaminate food and feed commodities (3, 36). A typical AF, AFB 1 for example, is produced by the following generally accepted pathway: acetate building blocks (also hexanoate)3anthrone-derivative polyketide3norsolorinic acid (NA)3averanfin3averufin3hydroxyversicolorine3versiconal hemiacetal acetate3versicolorin B3versicolorin A3sterig-matocystin3o-methylsterigmatocystin3AFB 1 (13,15,27,58). NA is polyketide derived and represents the first stable intermediate in AF biosynthesis (34,37). An unstable anthronederivative polyketide has been proposed as a hypothetical intermediate formed prior to NA, but this theoretical polyketide has not been isolated, presumably because it is rapidly oxidized to NA (27).Genes involved in the initial steps of the pathway are still unknown; their identification and characterization are essential to understanding the initiation of AF biosynthesis (15). In the initial steps of AF biosynthesis, there is predicted to be a polyketide synthase (PKS), but the enzyme has not been is...

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“…DNA was purified from A. flavus by the method of Horng et al (11). DNAs from A. flavus AF13 and the AF13 aflX knockout isolates were subjected to EcoRI, HindIII, or XhoI restriction enzyme digestion and separated by agarose gel electrophoresis, followed by vacuum blotting to Nytran Plus nylon membranes (Schleicher & Schuell, Inc., Keene, NH).…”

Section: Methodsmentioning

confidence: 99%

The Aflatoxin Biosynthesis Cluster Gene, aflX , Encodes an Oxidoreductase Involved in Conversion of Versicolorin A to Demethylsterigmatocystin

Cary

Ehrlich

Bland

et al. 2006

Appl Environ Microbiol

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Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion. A homolog of this gene, stcQ, is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated ϳ4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO (avfA). Some of domains in the protein are similar to those of epoxide hydrolases.The aflatoxins (AF), produced by Aspergillus species, are perhaps the most intensively studied polyketide metabolites (19)(20)(21). They are among the most potently toxic and carcinogenic compounds in nature (3). Biosynthesis of AF involves a set of coregulated genes that encode at least 27 proteins, including a Cys 6 Zn 2 -type pathway-specific transcription factor, a polyketide synthase, two dedicated fatty acid synthases, six cytochrome P450 monooxygenases, one esterase, two O-methyltransferases, two nonoxidative proteins, ten oxidoreductases, and two proteins for which there is little homology to known proteins in sequence databases (8,20).The roles of the proteins encoded by most of these genes in AF biosynthesis have been confirmed by either gene knockout or gene complementation studies (22). Conversion of versicolorin A (VA) to demethylsterigmatocystin (DMST) is predicted to involve multiple steps (Fig. 1). The probable sequence of steps has recently been clarified by analysis of precursor incorporation results (9, 10). The predicted steps involve oxidation of the anthraquinone moiety of VA, reductive deoxygenation of the A-ring, Baeyer-Villiger oxidation and rearrangement, and dehydration and decarboxylation. Enzymes previously shown to be involved in these steps are: a cytochrome P450 monooxygenase (VerA/StcS) (13, 14), an NADH-dependent deoxygenase (Ver-1/StcU) (16) with similarity to the melanin biosynthesis enzyme, tetrahydroxynaphthalene reductase (18), and a novel predicted metallo-oxidase, AflY (HypA/StcR) (6). The hypothetical pathway shown in Fig. 1 suggests that additional enzymes may be required to catalyze two additional steps in the conversion process: the epoxide ring-opening after VA oxidation and the dehydration/decarboxylation that follows the Baeyer-Vil...

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Development of a homologous transformation system for Aspergillus parasiticus with the gene encoding nitrate reductase

Cited by 57 publications

References 18 publications

Nonfunctionality of Aspergillus sojae aflR in a Strain of Aspergillus parasiticus with a Disrupted aflR Gene

Nonfunctionality of Aspergillus sojae aflR in a Strain of Aspergillus parasiticus with a Disrupted aflR Gene

Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus

The Aflatoxin Biosynthesis Cluster Gene, aflX , Encodes an Oxidoreductase Involved in Conversion of Versicolorin A to Demethylsterigmatocystin

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