Purification and Characterization of O-Methyltransferase I Involved in Conversion of Demethylsterigmatocystin to Sterigmatocystin and of Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin during Aflatoxin Biosynthesis

O-Methyltransferase I, which catalyzes conversions both of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and of dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST) during aflatoxin biosynthesis, was purified to apparent homogeneity from the cytosol fraction of the mycelia of Aspergillus parasiticus NIAH-26 through the following chromatography series: phenyl-Sepharose, DEAE-Sepharose, phenyl-Sepharose, Sephacryl S-300, and Matrex gel Green A. The apparent molecular mass was estimated at 150 kDa based on Sephacryl S-300 gel filtration chromatography, and the denaturing molecular mass was 43 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI of the enzyme was 4.4, and the optimal pH for activity was broad, from 6.5 to 9.0. In competition experiments using the purified enzyme, the formation of ST from DMST was suppressed when DHDMST was added to the reaction mixture and DHST was newly formed. These results indicate that DMST and DHDMST commonly serve as substrates for the enzyme. The K_m of the enzyme for DMST was 0.94 μM, and that for DHDMST was 2.5 μM. Interestingly, MT-I kinetics deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at a higher substrate concentration.     

Cloning and characterization of the O-methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis.

O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding O-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5' and 3' rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using Aspergillus parasiticus NIAH-26. A comparison of the genomic sequences with the cDNA of the dmtA region revealed that the coding region is interrupted by three short introns. The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5' flanking region of the stcP gene, suggesting that translation of the stcP gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5' and 3' flanking regions of the dmtA gene in which TAIL-PCR was used demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes. Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of the dmtA gene showed that a dmtA homolog is present in Aspergillus oryzae SYS-2.     

The Affinity Purification and Characterization of a Dehydrogenase from Aspergillus parasiticus Involved in Aflatoxin B1, Biosynthesis

Abstract

A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to ω-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140 000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25[ddot] to 35[ddot]C. The Km of the enzyme for NA and NADPH was determined to be 3.45 μM and 103 μM respectively.     

Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.     

Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in Aspergillus parasiticus

The nor-1 gene is involved in aflatoxin biosynthesis in Aspergillus parasiticus and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed in Escherichia coli converted the 1′ keto group of norsolorinic acid to the 1′ hydroxyl group of averantin in crude E. coli cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro.     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Dillapiol and Apiol as Specific Inhibitors of the Biosynthesis of Aflatoxin G1 in Aspergillus parasiticus

Dillapiol was isolated from the essential oil of dill as a specific inhibitor of aflatoxin G₁ production. It inhibited aflatoxin G₁ production by Aspergillus parasiticus with an IC₅₀ value of 0.15 μM without inhibiting aflatoxin B₁ production or fungal growth. Apiol and myristicin, congeners of dillapiol, showed similar activity with IC₅₀ values of 0.24 and 3.5 μM, respectively.     

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

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 ~4-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 Aspergillus parasiticus estA-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis

In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus. We deleted the estA gene from A. parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain. The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.     

Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-β-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

植物果聚糖合成酶基因克隆及特性分析

果聚糖(fructan)是蔗糖来源的果糖多聚体,果聚糖的类型常因聚合度的多寡、分支结构有无、相邻果糖基成键差异及葡萄糖位置不同而多种多样。果聚糖不仅是植物储存非结构性碳水化合物的形式之一,而且在干旱、低温等非生物胁迫中具有保护植物细胞免受伤害的作用。果聚糖作为一种益生素,对人体健康具有保健作用,有效减少或避免结肠癌、心血管疾病及骨质疏松等发病机率。本文就果聚糖的存在形式、生物合成代谢途径、果聚糖合成酶（fructan biosynthetic enzymes, FBEs）基因的克隆和转化等研究做一介绍,并对植物中FBEs结构特点进行了分析,同时对小麦中FBEs基因的拷贝数、染色体定位及亚细胞定位等研究进行了商榷,为从更多植物中分离FBEs基因,研究FBEs基因的作用方式和表达特性,以及利用转基因技术提高重要作物抗逆性奠定基础。     

Purification and Gene Cloning of a Dehydrogenase from Lactobacillus brevis That Catalyzes a Reaction Involved in Aflatoxin Biosynthesis

When 10 strains of lactic acid bacteria were incubated with 5′-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5′-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS–PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.     

Purification and characterization of mycoferritin from Aspergillus parasiticus (255)

As intracellular iron storage molecules, only hydroxymate type siderophores have been reported in ascomycetes and basidiomycetes. This is the first report documenting the presence of mycoferritin in ascomycetes. The fungus, Aspergillus parasiticus (255), is capable of producing mycoferritin only upon induction with iron in yeast extract sucrose (YES) medium. The same has been purified from Aspergillus sps by application of conventional biochemical techniques. The molecular mass, yield, iron and carbohydrate contents of the HPLC purified protein were 460kDa, 0.012mg/g of wet mycelia, 1.6% and 6.0%, respectively. The iron content was much lower than Mortierella alpina mycoferritin (17%). Native PAGE revealed the presence of trimeric and monomeric forms of ferritin. Subunit analysis by SDS-PAGE showed a single protein subunit of approximately 20kDa suggesting structural simplicity of the apoferritin shell. Variation in amino acid composition was noted upon comparison with ferritins of other species. Interestingly, no phenylalanine could be detected in the mycoferritin of Aspergillus sps. The acidic amino acid content was 1.5-1.6 fold higher than mammalian and fish ferritins. The spectral characteristics (UV/VIS and fluorescence) of mycoferritin were akin to equine spleen ferritin. However, circular dichroic spectra revealed a lower degree of helicity.     

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.     

Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus

We characterize the mating-type genes in Aspergillus flavus,Aspergillus parasiticus and Petromyces alliaceus. A single MAT1-1 or MAT1-2 gene was detected in the genomes of A. flavus and A. parasiticus, which is consistent with a potential heterothallic organization of MAT genes in these species. In contrast, the only known, functionally homothallic species in Aspergillus section Flavi, P. alliaceus, has tightly linked (<2kb) MAT1-1 and MAT1-2 genes, typical of other self-fertile homothallic euascomycetes. This is the first example of linked MAT genes within a homothallic species of Aspergillus. We tested the null hypothesis of no significant difference in the frequency of MAT1-1 and MAT1-2 in A. flavus and A. parasiticus sampled from a single peanut field in Georgia. For each species, mating-type frequencies were determined for the total population samples and for samples that were clone-corrected based on vegetative compatibility groups (VCGs) and aflatoxin gene cluster haplotypes. There was no significant difference in the frequency of the two mating types for A. flavus and A. parasiticus in either VCG or haplotype clone-corrected samples. The existence of both mating-type genes in equal proportions in A. flavus and A. parasiticus populations, coupled with their expression at the mRNA level and the high amino acid sequence identity of MAT1-1 (77%) and MAT1-2 (83%) with corresponding homologs in P. alliaceus, indicates the potential functionality of these genes and the possible existence of a sexual state in these agriculturally important species.