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1.
A patulin-negative mutant (J1) of Penicillium urticae (N.R.R.L. 2159A) was known to accumulate about 100mg per litre quantities of the 5,6-epoxygentisyl quinone, (-)-phyllostine and another metabolite (UIII). Both were derived from acetate and hence were polyketides. Purified UIII (m.p. 53 degrees C, [alpha](32) (D)+206 degrees , lambda(methanol) (max.) 240nm; epsilon 3806 litre.mol(-1).cm(-1)) was characterized as a partially reduced derivative of (-)-phyllostine and was found to be a diastereoisomer of the known phytotoxin, (+)-epoxydon. Hence its designation as (+)-iso- or epi-epoxydon. From (1)H n.m.r. and c.d. data the stereochemistry of the epoxide ring in (+)-isoepoxydon was determined to be identical with that in (+)-epoxydon (i.e. R,R) but the configuration of the secondary alcohol at C-4 was S rather than R as in (+)-epoxydon. Isoepoxydon (compound UIII) is therefore (4S,5R,6R)-5,6-epoxy-4-hydroxy-2-hydroxymethylcyclohex-2-en-1-one. The boat conformation in which the C-4 hydroxy group is axial is preferred. In the range of 1mm to 5mm, the antibiotic activity of (+)-isoepoxydon against Bacillus subtilis sp. was 56% of that obtained with patulin. Over a period of 1 to 3h, [(14)C]isoepoxydon was efficiently converted into patulin by a shake culture of the parent strain of P. urticae. The precursor relationship of isoepoxydon to patulin was confirmed by feeding unlabelled isoepoxydon (1mm) to a washed-cell suspension of a mutant (J2) in which, over a period of 3 to 5h, a better than 60% conversion into patulin was attained. The enzymic relationship between isoepoxydon and phyllostine and their positions in the late portion of the patulin biosynthetic pathway are discussed. 相似文献
2.
A patulin negative mutant (J1) of Penicillium urticae (NRRL 2159A) was found to accumulate large quantities (greater than 128 mg/L culture) of a reactive, photosensitive compound, which was isolated and identified as (-)-phyllostine (5,6-epoxygentisylquinone). This epoxyquinone possessed an antibiotic activity against Bacillus subtilis which was approximately 80% of that exhibited by patulin. In separate in vivo feeding experiments, [2-14C]acetate and [G-3H]gentisaldehyde were readily incorporated into phyllostine by mutant J1 and [14C]phyllostine was incorporated into patulin by the parent strain (NRRL 2159A). When fed to a washed-cell suspension of a second patulin negative mutant (J2) which produced gentisaldehyde but not phyllostine, unlabeled phyllostine was efficiently converted to patulin in yields of 33, 56, and 92% after 30 min, 1 and 5 h, respectively. The role of phyllostine as an intermediate of a new post-gentisaldehyde portion of the patulin biosynthetic pathway is discussed. 相似文献
3.
Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence
of the isoepoxydon dehydrogenase ( idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce
the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA
(ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design
of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi.
The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged. 相似文献
4.
Submerged cultures of Penicillium urticae (NRRL 2159A) produced the antibiotics patulin and griseofulvin when grown in a glucose-nitrate medium. A high concentration of calcium (i.e., 68 mM) inhibited the production of both antibiotics while stimulating conidiogenesis. Conidial mutants that were defective in an early stage of conidiogenesis produced markedly less patulin, even under growth conditions that favored secondary metabolism. A mutant which lacked the ability to produce the patulin pathway metabolites m-cresol, toluquinol, m-hydroxybenzyl-alcohol, m-hydroxybenzaldehyde, gentisaldehyde, gentisyl alcohol, gentisic acid and patulin, as well as the pathway enzyme m-hydroxybenzyl-alcohol dehydrogenase, still produced yields of conidia that were equivalent to or greater than those of the parent strain. Other mutants which were blocked at later steps of the patulin pathway also produced conidia. These results indicate that patulin and the other related secondary metabolites noted above are not a prerequisite to conidiogenesis in P. urticae. Environmental and developmental factors such as calcium levels and conidiogenesis do, however, indirectly affect the production of patulin pathway metabolites. 相似文献
5.
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase family, catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. A relatively conserved threonine residue corresponding to threonine 11 of 15-PGDH is proposed to be involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine the important role of this residue. Threonine 11 was changed to alanine (T11A), cysteine (T11C), serine (T11S) or tyrosine (T11Y) and the mutant proteins were expressed in E. coli. Western-blot analysis showed that the expression levels of mutant proteins were comparable to that of the wild-type enzyme. Mutants T11A, T11C and T11Y were found to be inactive. Mutant T11S still retained substantial activity and the K(m) value for prostaglandin E(2) (PGE(2)) was similar to the wild-type enzyme; however, the K(m) value for NAD(+) was increased over 23-fold. These results suggest that threonine 11 may be involved in the interaction with NAD(+) either directly or indirectly and contributes to the full catalytic activity of 15-PGDH. 相似文献
6.
The effect of trace metal nutrition on the functioning of the patulin biosynthetic pathway in submerged cultures of Penicillium urticae (NRRL 2159A) was examined by both chromatographic and enzymological means. Comprehensive metal ion analysis showed generally low levels of contaminating metal ions in media components. Of eight metal ions examined, only manganese strongly influenced secondary metabolite production. In control cultures or cultures deficient in calcium, iron, cobalt, copper, zinc, or molybdenum, pathway metabolites appeared in the medium at about 25 h after inoculation. The first pathway-specific metabolite, 6-methylsalicylic acid, accumulated only transiently before being converted to patulin whose concentration steadily increased. In manganese-deficient cultures, however, 6-methylsalicylic acid continued to accumulate, with only minor amounts of patulin being produced. Additionally, a marker enzyme for the pathway showed only 0-20% of control activity. Clear dose responses (patulin versus manganese) were found in different media, with no effect on growth yield. Addition of manganese to depleted cultures at 18, 26, or 36 h resulted in increasing marker enzyme activity and patulin concentrations. It is concluded that manganese exerts a specific, positive effect on patulin biosynthesis and may in some way control the section of the patulin pathway occurring after 6-methylsalicylic acid. 相似文献
7.
Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols. 相似文献
9.
Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats. 相似文献
10.
A crude extract that catalyzes the epoxidation of toluquinol and gentisyl alcohol was isolated from cultures of Penicillium patulum. About 60% of the activity sedimented from crude extract upon centrifugation at 105,000g for 2 h, and at 30,000g for 30 min after precipitation with 30% ammonium sulfate and resuspension in buffer. The quinone epoxide phyllostine, a product of gentisyl alcohol epoxidation, has previously been shown to be an intermediate in the biosynthesis of patulin [Sekiquchi, J., & Gaucher, G. M. (1978) Biochemistry 17, 1785-1791] and was shown to be further converted to neopatulin by the extract. The epoxide product of toluquinol, desoxyphyllostine (2-methyl-5,6-epoxy-1,4-benzoquinone), has not been reported previously from fungal cultures. Its structure was confirmed by GC-mass spectrometry and proton and 13C NMR. Its CD spectrum showed the same shape and signs as that of phyllostine, indicating that it too is an enzymatic product with a similar absolute configuration. Whereas chemical epoxidation of toluquinone and gentisyl quinone occurs with hydrogen peroxide, the enzymatic epoxidation utilized oxygen and the hydroquinone. The epoxidation was inhibited by 1,10-phenanthroline, EDTA, and p-(chloromercuri)benzenesulfonic acid and by degassing with nitrogen, but no inhibition was observed with KCN, catalase, or CO. The apparent Km's were similar for the two substrates (0.17 mM for toluquinol, 0.24 mM for gentisyl alcohol), with both substrates showing inhibition at 1.0 mM. The rate of desoxyphyllostine formation was more than 10 times that of phyllostine formation at equivalent substrate concentrations. Gentisaldehyde was not a substrate for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
The taxonomy of the penicillia is unstable particularly in the important antibiotic and mycotoxin-producing subgenus Penicillium. There are difficulties relating identifications to mycotoxin production. Also, the validity of dual nomenclature for pleomorphic fungi is under discussion increasingly. Patulin is an important mycotoxin produced by various fungi and has strict limits in the European Union. The mycotoxin and/or the isoepoxydon dehydrogenase (IDH) gene of the metabolic pathway have been assessed in 318 strains predominately of subgenus Penicillium. These data were used to classify the isolates. Subgenus Penicillium contained most of the IDH and patulin positives. The species and varieties in subgenus Penicillium which were associated with patulin detection can be reduced to one name, viz. Penicillium Pen p+ (p = patulin). This has been extended to other mycotoxin producing penicillia to indicate the scope of the scheme. The classification will lead to the number of taxa being reduced, while avoiding species names and hence dual nomenclature. Culture independent analysis of environmental samples is mentioned. The scheme could be used with advantage for other fungi. 相似文献
12.
This study evaluates the specificity of PCR isoepoxydon dehydrogenase (idh) primers on fungi associated with patulin production. The DNAs of 93 strains were extracted and analysed by PCR using primers of the idh gene of patulin biosynthesis. A single band at 620 bp was obtained on 17% of the analysed strains. Different molecular weight amplicons were observed in other strains. These were employed as binary characters for numerical analysis to obtain a dendrogram. Clusters were observed, which corresponded to morphological identifications in some cases. Amplicons at 400 and/or 500 bp were related to patulin non-detection for strains, whereas a 450 bp amplicon was associated with some Aspergillus and both of the Byssochlamys nivea strains tested. Hence, the idh primers are not specific for the gene and provide other amplicon products in other species. These results were useful providing (a) profiles of DNA to identify and classify fungi and (b) insights into patulin production. The DNA profiles in this study may be useful for determining patulin producing fungi. Obtaining multiple bands in culture-independent PCR of environmental samples by using the primers could indicate that more than one species is present.
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