A manganese requirement for patulin biosynthesis by cultures of Penicillium urticae

Antibiotic production by submerged cultures of Penicillium urticae required manganese supplementation of the media. Thus, manganese supplementation (152 M) allowed accumulation of patulin to high concentrations (2 mol/mL), whereas manganese deficiency (1.53 M) resulted in the accumulation to similar levels of the first committed pathway intermediate, methyl-salicylic acid, without significant patulin accumulation. Preliminary studies suggest that a similar manganese effect may occur in other fungal species.     

Isoepoxydon, a new metabolite of the patulin pathway in Penicillium urticae

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.     

Identification of phyllostine as an intermediate of the patulin pathway in Penicillium urticae.

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.     

Manganese and antibiotic biosynthesis. I. A specific manganese requirement for patulin production in Penicillium urticae

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.     

Manganese and antibiotic biosynthesis. II. Cellular levels of manganese during the transition to patulin production in Penicillium urticae

The radionuclide 54MnCl2 was used to examine the cellular manganese content of submerged cultures of Penicillium urticae NRRL 2159A. Liquid-scintillation spectroscopy allowed sensitive detection of isotopic manganese in both normally supplemented and manganese-deficient cultures. The cellular manganese content in supplemented cultures showed three distinct phases, including a period of uptake that coincided with the time of transition to antibiotic biosynthesis. Such an uptake was not seen for manganese-deficient cultures, but addition of normal quantities of unlabelled manganese to the media appeared to stimulate uptake. Preliminary characterization shows this manganese uptake is not inhibited by other metal ions, does not require metabolic energy or a protein component, but is disrupted by changes in incubation temperature. The significance of these observations is discussed in the light of recent work on the requirement for manganese for antibiotic biosynthesis in this organism.     

In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.     

Penicillium urticae Bainier enumeration in soils

Summary A dilution plating method estimatedPenicillium urticae Bainier numbers in soil. This method, which used an agar layering technique and a cyclic incubation of 8 hours at room temperature (about 25°C) and 16 hours at 5°C, permitted the differential growth and sporulation in favor ofP. urticae B. over other common soil fungi.Procedures of extraction, paper chromatography, infrared analysis, and bioassay assayed for accumulated patulin. A combination of these methods routinely estimatedP. urticae B. numbers in soils and authenticated patulin production by selected isolates.Contribution from the Northern Plains Branch, Soil and Water Conservation Research Division, Agricultural Research Service, USDA, in cooperation with the Nebraska Agricultural Experiment Station, Lincoln. Published as Paper No. 2275, Journal Series, Nebraska Agricultural Experiment Station.Soil Scientist, USDA, Grand Junction, Colorado (formerly Chemist, USDA, Lincoln, Nebraska); and Microbiologist, USDA, Lincoln, Nebraska, respectively.     

Conidiogenesis and secondary metabolism in Penicillium urticae.

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.     

Growth and patulin formation byPenicillium urticae Bainier in pure and mixed cultures

Penicillium urticae Bainier synthesized patulin in potato-dextrose medium at temperatures ranging from 5 to 30°C. Maximum patulin yield was 2700 μg/ml of culture fluid in 14 days at 25°C. Two distinctive intervals affected patulin formation: 15 to 20°C and 30 to 35°C, the former favorable and the latter detrimental. An incubation period of 11 to 14 days made a nonsterile mixture of weathered wheat straw and soil a favorable medium for patulin formation. Autoclaved weathered wheat straw, inoculated withP. urticae alone, or in combination withTrichoderma sp., was a medium comparable to nonsterile, incubated weathered wheat straw in soil. Both carbon source and accessory growth factors were important for patulin formation. Of seven media tested, potato-dextrose was superior to potatodextrose supplemented with 70 ppm Zn-ions and 16 ppm Fe-ions, potatosucrose, Raulin-Thom, autoclaved weathered wheat straw in pure culture, weathered wheat straw in nonsterile soil, and autoclaved weathered wheat straw in mixed culture, in that order. Patulin production ranged from 337.5 to 0.2 mg/g of C in the medium. Contribution from the Northern Plains Branch, Soil and Water Conservation Research Division, Agricultural Research Service, U.S. Department of Agriculture, in cooperation with the Nebraska Agricultural Experiment Station, Lincoln. Published as Paper No.2621, Journal Series, Nebraska Agricultural Experiment Station.     

Inhibition of growth and patulin synthesis in Penicillium expansum by potassium sorbate and sodium propionate in culture.

Potassium sorbate and sodium propionate brought about a marked inhibition in the growth of Penicillium expansum and a proportionally greater inhibition in the synthesis of patulin by the mold. At inhibitor concentrations used commercially in bakery products, propionate inhibited growth less efficiently than sorbate did but was a more effective inhibitor of patulin synthesis.     

Regulation of beta-1,3-glucanase synthesis in Penicillium italicum.

The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.     

Patulin Production by Penicillium urticae Bainier in Batch Culture

A still, batch-culture method, with potato dextrose medium and Penicillium urticae Bainier, produced patulin yields of 1.2 to 1.7 g/liter of medium. Incubation was at 25 C for 14 days. Ethyl acetate extraction of condensed culture filtrate and drying with anhydrous MgSO₄, followed by solvent change to dry ethyl ether and purification on alumina (pH 4.5), produced pure crystalline patulin. The use of 2-liter, round-bottom flasks and a rotating vacuum evaporator provided versatile equipment and easy manipulation in the operations. Soil from wheat fields provided a convenient natural P. urticae source. Potato dextrose medium was superior to potato sucrose or Raulin-Thom media.     

Produciton and biological activity of patulin and citrinin from Penicillium expansum

Penicillium expansum isolated from meat and apples produced both patulin and citrinin. Toxin identity was confirmed by spectroscopic and physical methods. The mean lethal dose in chicken embryos was determined for toxins administered both singly and in various ratios. Data from simultaneous administration of mycotoxin combinations plotted as isobolograms showed and additive effect. Both toxins were teratogenic in chicken embryos.     

The isoepoxydon dehydrogenase gene of the patulin metabolic pathway differs for Penicillium griseofulvum and Penicillium expansum

Purified DNA from isolates of Penicillium griseofulvum and P. expansum was used as a template to amplify a 600-bp fragment of the isoepoxydon dehydrogenase (idh) gene of the patulin biosynthetic pathway. Primer pairs designed from the P. griseofulvum gene (GenBank accession AF006680) to amplify specific regions of the idh gene yielded similar-sized bands for all strains. Asymmetrical amplification produced DNA products for sequencing and DNA sequences were translated to produce the corresponding amino acid sequences. After removal of two introns present in the region sequenced, amino acid sequences were compared. There were 12 amino acid differences between P. expansum and P. griseofulvum in the coding region. The differences correlated with the amount of patulin previously produced in culture, with strains of P. griseofulvum producing the greatest amounts of patulin.     

Effect of preservatives on patulin production by Penicillium expansum.

Penicillium expansum has been grown on Capek-Dox medium using glucose and fructose as carbon source. Preservatives used in fruit processing and introduced in the medium were sorbic acid, formic acid, benzoic acid, SO2 and saccharose. Sulphur dioxide had a most inhibitory effect on mycelium growth and patulin production, formic acid concentration of 0.025% increased the amount of patulin by about 30% as compared to the culture with no preservatives. However its higher concentrations inhibited synthesis of this mycotoxin. Sorbic acid concentration of 0.1% stimulated the fungus strains examined in patulin synthesis but its highest amounts were detected using 0.0125% benzoic acid increased patulin secretion from 8 to 50% as compared to the control, depending on the strain examined. Saccharose concentration up to 50% clearly decreased patulin content in the medium until its total disappearance.     

Produciton and biological activity of patulin and citrinin from Penicillium expansum.

Penicillium expansum isolated from meat and apples produced both patulin and citrinin. Toxin identity was confirmed by spectroscopic and physical methods. The mean lethal dose in chicken embryos was determined for toxins administered both singly and in various ratios. Data from simultaneous administration of mycotoxin combinations plotted as isobolograms showed and additive effect. Both toxins were teratogenic in chicken embryos.