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Biosynthesis of ochratoxins by Aspergillus ochraceus
Phytochemistry 58 (2001) 709–716 www.elsevier.com/locate/phytochem
Biosynthesis of ochratoxins by Aspergillus ochraceus Jonathan P. Harris, Peter G. Mantle* Biochemistry Department, Imperial College of Science, Technology and Medicine, London, SW7 2AY, UK Received 19 January 2001; received in revised form 5 July 2001
Abstract Shaken liquid fermentation of an isolate of Aspergillus ochraceus showed growth-associated production of ochratoxins A and B, followed by production of a related polyketide diaporthin. Later, between 150 and 250 h, mellein accumulated transitorily. In contrast, shaken solid substrate (shredded wheat) fermentation over 14 days produced mainly ochratoxins A and B (ratio ca. 5:1) in very high yield (up to 10 mg/g). In these systems experiments with 14C-labelled precursors and putative intermediates revealed temporal separation of early and late stages of the ochratoxin biosynthetic pathway, but did not support an intermediary role for mellein. The pentaketide intermediate ochratoxin b was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin a was only biotransformed significantly (4.85%) into ochratoxin A, indicating that chlorination is mainly a penultimate biosynthetic step in the biosynthesis of ochratoxin A. This was supported by poor (1.5%) conversion of radiolabelled ochratoxin B into ochratoxin A. Experiments implied that some ochratoxin B may arise by dechlorination of ochratoxin A. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus ochraceus; Pulse-radiolabeling; Shaken solid substrate fermentation; Ochratoxin A; Ochratoxin B; Ochratoxin a; Ochratoxin b; Mellein; Ethionine
1. Introduction The mycotoxin ochratoxin A (OTA, 1) (Fig. 1), first obtained from a South African Aspergillus ochraceus isolate (van der Merwe et al., 1965a), consists of a dihydroisocoumarin moiety (the pentaketide-derived ochratoxin a, 3; Fig. 2) linked through the carboxyl group to phenylalanine. Corresponding des-chloro analogues are ochratoxins B (OTB, 2) and b (4). Unlike ochratoxin a, ochratoxin b and mellein (5) have both been isolated from ochratoxinogenic A. ochraceus fermentations (Moore et al., 1972; Delgadillo, 1986). 14 C-labelled precursor feeding experiments and subsequent chemical degradation showed that phenylalanine was incorporated into OTA, whereas ochratoxin a was constructed from five acetate units with a one carbon addition at C-7 from methionine (Ferriera and Pitout, 1969; Searcy et al., 1969; Steyn et al., 1970). A crude cell-free enzyme preparation (OTA synthetase, requiring ATP and Mg++ ions) catalysed the linking of * Corresponding author. Tel.: +44-207-594-5245; fAx: +44-207225-0960. E-mail address: [email protected] (P.G. Mantle).
ochratoxin a with phenylalanine (Ferriera and Pitout, 1969). Wei et al. (1971) demonstrated incorporation of 36 Cl into OTA. The methylation inhibitor ethionine completely inhibited OTA production (Yamazaki et al. 1971). The 13C NMR spectrum of OTA was fully assigned by de Jesus et al. (1980); ochratoxinogenic fungal cultures fed [1-13C] or [1, 2-13C2]acetate showed enhanced signals and 13C–13C coupling evidence supporting a pentaketide folding pattern similar to that in the biosynthesis of citrinin and mellein. Huff and Hamilton (1979), recognising the lack of research in the pathway of OTA biosynthesis, proposed a scheme, although this unfortunately ignored the ubiquitous OTB. Subsequently, the ochratoxin polyketide synthase was reported to produce mellein; Abell et al., (1982) showed that there was no keto-enol tautomerism, but an unfunctionalised double-bond between C-6 and C-7 in the mellein pentaketide chain. Also, the carbonyl at C-3 may have been reduced to a hydroxyl prior to folding (Abell et al., 1983). The requisite methylation step was thought to occur after the production of mellein although the subsequent putative intermediates 7methylmellein, 7-methoxymellein and 7-formylmellein
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00316-8
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limit of 5 mg/kg (5 ppb) in commodities for human food. It is timely, therefore, to contribute to the understanding of some fermentation dynamics of A. ochraceus, as one of the fungi involved in commodity spoilage, and of some later steps in the biosynthesis of ochratoxins.
2. Results and discussion 2.1. Experiments in shaken liquid fermentation
Fig. 1. Structures of the Aspergillus ochraceus mycotoxins ochratoxins A and B.
Fig. 2. Structures of the dihydroisocoumarins ochratoxins a and b and mellein.
have not been isolated from fungal fermentation producing ochratoxins. Chlorination of ochratoxin b by a chloroperoxidase was reasoned as occurring prior to joining the aromatic nucleus to phenylalanine via an acyl activated phosphoochratoxin a reacting with phenylalanine with its carboxy group protected by ethyl esterification. This reaction would result in ochratoxin C which could be transesterified to the OTA methyl ester, both of which OTA derivatives have been isolated from A. ochraceus cultures (van der Merwe et al. 1965b; Steyn and Holzapfel, 1967). Alternatively, ochratoxin C might be hydrolysed directly to OTA. OTA, as a nephrotoxin and a carcinogen, is currently the subject of proposed EC legislation to place an upper
2.1.1. Culture dynamics In potato dextrose broth, accumulation of biomass was complete within 3–4 days and was closely associated with production of ochratoxins (Fig. 3) (also noted by Steyn et al., 1970; Wei et al., 1971). There were also second and third temporal phases of secondary metabolite occurrence measured concerning the polyketides diaporthin (6; Fig. 4) and mellein. Throughout the phase of ochratoxin occurrence there was a ratio of A:B of approximately 3:1. Diaporthin was also accompanied by approximately half the amount of orthosporin (de-O-methyl diaporthin; Harris and Mantle, 2001). The transitory appearance of mellein might plausibly be interpreted as reflecting cessation of involvement of this putative intermediate in ochratoxin biosynthesis after growth-associated production of the phenylalanine moiety declined. Alternatively, the observed temporal sequence of metabolites simply reflected progressive decline in biosynthetic complexity from ochratoxins (requiring phenylalanine, methionine, acetate and chlorine) to mellein which is solely derived from acetate. 2.1.2. Effect of ethionine The only marked effect of the methylation inhibitor ethionine on accumulation of ochratoxins in submerged fermentation was on yield of OTA when added during early idiophase at 32 h (Table 1). This is consistent with methylation of the pentaketide occurring early in biosynthesis. The less obvious or nil effect on excreted OTB would be consistent with some of this arising by dechlorination of OTA. 2.1.3. Radioactivity experiment Radiolabel from [1-14C]acetate was incorporated into OTA when added as early as only 8 h into the fermentation; the finding demonstrated the close association of ochratoxin biosynthesis with replicatory growth (Table 2). The optimum time of addition of the polyketide precursor to achieve maximum specific radioactivity in OTA was at 22.5 h, consistent with a principle of maximum rate of isocoumarin biosynthesis preceding prominent occurrence of OTA in broth (Fig. 3). Reinterpretation of the data of Lillehoj et al.
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Fig. 3. Dry cell weight (DCW), diaporthin ([Diap]), ochratoxin A ([OTA]), ochratoxin B ([OTB]) and mellein ([Mellein]) concentration versus time for an A. ochraceus fermentation in potato dextrose broth. Table 2 Effect of adding [1-14C]acetate (5 mCi) to potato dextrose broth fermentations at different stages on the amount and specific radioactivity of [14C]ochratoxin A recovered and analysed 80 h after inoculation
Fig. 4. Structure of diaporthin (6).
Table 1 Effect of adding ethionine (50 mg) at different fermentation stages in potato dextrose broth on the concentrations of ochratoxins A and B measured 80 h after inoculation
Addition time (h)
Ochratoxin A (mg)
Specific radioactivity (dpm/mg)
8 22.5 32 50
23 143 143 152
6 37 18 15
2.2. Experiments in shaken solid substrate fermentation
ochraceus growth in shaken shredded wheat substrate (Fig. 5). Accumulation of OTA and OTB closely followed fungal growth and was near maximum after 2 weeks; a 5:1 ratio up to day 9 diverged later in favour of OTB, possibly reflecting limitation of substrate chlorine ions for OTA formation (Stander et al., 2000). Yields of 10 mg OTA/g substrate have been achieved. If ergosterol were even as much as 1% of A. ochraceus mycelial biomass, OTA would become 1/10th of fungal biomass; this is probably a conservative estimate. Mellein, diaporthin and orthosporin were absent or barely detected throughout this two-week fermentation. Contentious aspects of ochratoxin biosynthesis were therefore explored in this unusual medium in which secondary metabolism in A. ochraceus was highly focused on the ochratoxin pathway.
2.2.1. Culture dynamics Ergosterol, as a measure of fungal mycelium in the substrate, increased linearly through days 4–8 of A.
2.2.2. Radioactivity experiments The magnitude of the relative incorporations of the three radioactive biosynthetic precursors into OTA and
Ethionine addition times (h)
[Ochratoxin A] (mg/l)
[Ochratoxin B] (mg/l)
32 50 56 72 Control
2.1 4.2 3.7 4.5 4.8
1.2 1.3 1.3 1.3 1.4
(1978) reveals evidence of a similar temporal separation of steps in ochratoxin biosynthesis.
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Fig. 5. Changes in ergosterol ([Ergo]), ochratoxin A ([OTA]) and ocratoxin B ([OTB]) concentraion during 2 weeks of an Aspergillus ochraceus solid substrate fermentation.
OTB, when added to fermentations on days 3, 4, 5 or 6 (Figs. 6 and 7), showed that the experimental system was well suited to biosynthetic study. The data also demonstrated temporal separation of optima for isocoumarin biosynthesis (day 4 for OTA; days 4–5 for OTB) and addition of the phenylalanine moiety (day 5 for OTA; days 5–6 for OTB). Notably, 50% of the l[U-14C]phenylalanine added at 5 days was incorporated into OTA and OTB. Relative incorporations of the putative intermediates ochratoxins a and b and mellein (Table 3) indicated a strong preferential role of a into A, but similar strong involvement of b into both A and B as subsequently isolated. 14C from mellein occurred in much smaller amount in ochratoxins, which may only reflect indirect incorporation of the radiolabel from mellein catabolism. To suggest that the rather negative experimental result arose from poor uptake of added mellein seems weak when added ochratoxins a and b, both only having an additional carboxyl group, were incorporated so extensively into OTA and OTB. Mellein was only a transitory product in shaken liquid culture (Fig. 3) and therefore A. ochraceus is well able to degrade it; no trace of mellein was found in the fermentation fed [14C]mellein. Indeed, the same putative indirect incorporation may explain the small amount of radiolabel in OTB after feeding with ochratoxin a. Autoradiography of chromatograms of neutral and acidic components of extracts of cultures fed ochratoxin a showed some 14C in neutral non-polar components, in
Fig. 6. The % incorporation of 5 mCi of either [1-14C]acetate, [methyl-14C]methionine or [U-14C]phenylalanine into [14C]ochratoxin A during 5 h after adding to Aspergillus ochraceus solid substrate fermentations on days 3, 4, 5 and 6.
addition to qualitative confirmation of incorporation into ochratoxins. This supports the interpretation of only indirect incorporation from ochratoxin a to OTB. Conversely, there was no autoradiographic evidence of biotransformations in the fermentation given ochratoxin b other than into OTA and OTB. In the fermentation given [14C]mellein there were no particular locations of radiolabel in any fraction of extract, supporting the notion of its rather rapid and extensive catabolism. In following the fate of dual-labeled OTA and OTB in shaken shredded wheat culture, more than 40% of the added OTA was recovered in the form as added (Table 4). However, an amount equivalent to ca.10% was present as OTB although with the 3H:14C ratio being slightly different. If this is a real change it might imply that the phenylalanine moiety can be transferred to OTB more readily than can the dihydroisocoumarin moiety, which would have to be dechlorinated. Very little (approx. 6.5%) of the added dual-labeled OTB was recovered as such with a similar 3H:14C ratio and even less was incorporated into OTA. Thus, experimental change of doublylabelled OTA into OTB 5 h after it was added to a fermentation in early idiophase (Table 4) was the most significant interconversion observed. Generally maintained
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Fig. 7. The % incorporation of 5 mCi of either [1-14C]acetate, [methyl-14C]methionine or [U-14C]phenylalanine into [14C]ochratoxin B during 5 h after adding to Aspergillus ochraceus solid substrate fermentations on days 3, 4, 5 and 6.
Table 3 The % incorporation of 0.13 mCi [14C]ochratoxin a, 0.1 mCi [14C] ochratoxin b or 0.096 mCi [14C]mellein into [14C]ochratoxins A and B during 5 h of solid substrate fermentation at the 4 day stage
Ochratoxin A Ochratoxin B
3
[14C]ochratoxin a
[14C]ochratoxin b
[14C]mellein
4.85 0.47
14.32 18.98
0.56 0.68
H:14C ratios confirmed the validity of most of the transformations observed. Correct interpretation of the findings depends on the extent of cellular uptake of the compounds administered; conversion of OTA to OTB as a catabolic step could be extracellular, but chlorination of OTB to OTA probably requires cellular uptake. If there is not an uptake mechanism for OTB, this could impose a limit on experimentation. It is concluded from all the experiments that chlorination of both ochratoxin b and OTB can give rise to OTA. However, there was no evidence for the intermediate role of the ester ochratoxin C proposed by Huff and Hamilton (1979), presumably interpreted as having a role in protecting the phenylalanine carboxyl during the last biosynthetic steps. These results suggest that one, possibly dominant,
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biosynthetic pathway involves ochratoxins b!a!A, with also a branch for ochratoxins b!B. An alternative pathway might be ochratoxins b!B!A but, if ochratoxin a is not directly involved, it is difficult to explain the present demonstration that it can occur naturally. However, mellein seems not to be involved as a precursor of either pathway. The discovery of 5-methyl-, 5formyl- and 5-carboxymellein in culture broth of Nummulariella marginata (Whalley and Edwards, 1985) provides a precedent for an oxidation system for a substituted methyl group, yet if such compounds are stable enough to be isolated from fermentation broth it is questioned why 7-methyl- and 7-formylmellein have not, so far, been discovered. Results of the second duallabeling experiment complemented the first (Table 4). Over half the added OTA was recovered and only 3% was incorporated into OTB although with a slightly different radionuclide ratio. Autoradiography of culture extract chromatograms showed no 14C in neutral fractions, in contrast to results from experiments with [14C] ochratoxins a and b and mellein. Where [14C] OTA or OTB was added, radioactivity was only obvious in the unchanged ochratoxin corresponding to what had been added. Thus, although it was shown that OTA can be converted into OTB nonspecifically, there was no convincing evidence whether or not specific biotransformation of OTB into OTA had occurred. Since the dual-labelled experiments did not achieve the high percentage incorporations of the experiments in which ochratoxins a and b were administered, it could be argued that the inter-conversion of OTA and OTB, although shown to occur, is not as important in the biosynthetic pathway as the reactions involving ochratoxins a and b.
3. Experimental 3.1. Fungi Two isolates of A. ochraceus were used; D2306 for its high yield of ochratoxins (Connole et al. 1981; as also used since by Tapia and Seawright, 1984, Stander et al., 2000; Stoev et al., 2000) and KBf for its production of mellein (though not producing ochratoxins; Mantle and McHugh, 1993). The fungi were maintained on potato dextrose agar (Difco). 3.2. Culture conditions Submerged liquid culture was in potato dextrose broth (Difco), 100 ml in 500 ml Erlenmeyer flasks, inoculated with spores to give 2–4106 spores ml1 and incubated on an orbital shaker (200 rpm, 10 cm eccentric throw), at 28 C for isolate D2306 and 22 C for isolate KBf.
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Table 4 The % incorporation and 3H:14C ratio of ochratoxins A and B recovered after adding dual-labelled ochratoxins A and B to solid substrate fermentations % Incorporation
3
Ochratoxin A added for 5 h on day 5 3 H Ochratoxin A 14 C 3 Ochratoxin B H 14 C
43.2 41.5 12.1 8.2
1:0.47
Ochratoxin B added for 5 h on day 5 3 H Ohratoxin A 14 C 3 Ochratoxin B H 14 C
1.4 1.5 6.7 6.4
1:0.43
Experiment
Isotope
Ochratoxin A added on day 6 and incubated overnight 3 H 58.3 Ochratoxin A 14 C 57.5 3 Ochratoxin B H 3.3 14 C 2
H:14C
on day 4, were each hydrolysed in 50 ml 6 M HCl by reflux for 18 h. The cooled mixture was extracted with EtOAc and ochratoxins a and b were purified by HPLC (see below) detected at 323 nm (retention time, Rt, 10 and 6 min, respectively, at 8.4 ml min1). 3.5. Biosynthetic preparation of [14C]mellein
1:0.48
Sodium [1-14C]acetate (10 mCi) was added to a 37 h liquid fermentation flask culture of isolate KBf and the fermentation continued for 1 h. [14C] Mellein (2.22 mg) was obtained by extraction of culture filtrate with EtOAc followed by C18 SepPak and HPLC purification (see below). The measured radioactivity (1.67 mCi) represented 16.7% incorporation of radiolabel from [1-14C]acetate.
1:0.29
3.6. Source of authentic compounds
1:0.33
1:0.37
Shaken solid substrate fermentation in Erlenmeyer flasks was developed from the principle first devised for aflatoxins (Shotwell et al., 1966), then applied to OTA (Hesseltine, 1972) and scaled up to rotating barrel fermenters (Lindenfelser and Ciegler, 1975). Shredded wheat breakfast cereal (Cereal Partners UK, Welwyn Garden City), 40 g per flask, was autoclaved, moistened to an initial moisture content of approximately 35% with sterile water (16 ml) and incubated on the orbital shaker at 28 C. Sterile water was also added at 24 and 48 h (4 and 2 ml, respectively). Water evaporation and metabolic dissimilation of substrate during the subsequent 2 weeks of shaken fermentation resulted in approximately 35 g of moist chocolate-coloured granules in each flask. Some small-scale fermentations were performed as 2 g of shredded wheat in 50 ml flasks for radiochemical experiments. 3.3. Biosynthetic preparation of dual-labeled OTA and OTB After 4 days in standard shredded wheat culture, 2 g material was transferred to a 50 ml flask and given 5 mCi each of [methyl-14C]methionine and l-[2, 3, 4, 5, 6-3H]phenylalanine. Shaken incubation continued for 5 h and the culture was extracted for OTA (3.4 mg, 0.15 mCi 3H:0.07 mCi 14C, ratio 3H:14C 1: 0.47) and OTB (1.5 mg, 0.29 mCi 3H:0.11 mCi 14C, ratio 3H:14C 1: 0.38). Radioactivity from phenylalanine was incorporated about twice as efficiently as that from methionine. 3.4. Preparation of 14C labeled ochratoxins and OTA (1.16 mg, 0.21 mCi) and OTB (1.44 mg, 0.14 mCi), from cultures fed [methyl-14C]methionine for 5 h
OTA and OTB were isolated from solid substrates (2 g) by extraction in 100 ml EtOAc:0.01 M H3PO4, 9:1 (v:v), partition into aqueous NaHCO3 (3%), acidified and back extracted into EtOAc. Residues were taken up in MeOH. OTA and OTB were purified by preparative HPLC (see later). Ochratoxins a and b were synthesized by the acid hydrolysis of 1 mg OTA or OTB, respectively, followed by preparative HPLC (see later). Mellein was obtained from 1 week potato dextrose broth culture of isolate KBf. The filtered broth, adjusted to pH 3, was extracted with EtOAc (100 ml) and the organic phase partitioned against a 3% aqueous NaHCO3 solution to remove acidic metabolites. The organic extract was taken to dryness. The solutes, dissolved in MeOH, were processed by HPLC (below; detection at 239 nm, Rt 30 min). Combined eluates corresponding to mellein were mixed with an equal volume of H2O and passed through a C18 SepPak classic cartridge pre-conditioned first with MeOH (20 ml) and then with H2O (20 ml). Mellein was eluted in EtOH in the 2nd and 3rd 0.5 ml fraction. Diaporthin and orthosporin were available from other studies (Harris and Mantle, 2001). All compounds were investigated by HR-MS, and linked scan EIMS (B/E and B2/E) to interpret fragmentation. The EI mass spectra of OTA and OTB differ by the chlorine or proton substituent at C5, respectively. Molecular ions are very weak, as are those corresponding to losses of H2O or CO2. Therefore, use of EIMS for confirming OTA relies on the two important fragment ions, in this case m/z 255 and 239 (base peak). These represent, respectively, the isocoumarin moiety still with the amino nitrogen of phenylalanine and the isocoumarin alone. Principles of further EI fragmentation of the latter, with a carboxyl group reconstituted at C10, are illustrated by ochratoxin a. It is helpful to have a rationale for occurrence of fragment ions in EIMS at
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least for diagnostic purposes and this has not been available before. From accurate mass measurements and linked-scan analysis of ochratoxins a and b, and of mellein, rationalised MS fragmentation [Scheme 1, extending the data of Delgadillo (1986)] showed considerable homology between ochratoxins a and b. Also, the composition of ion m/z 149 in ochratoxin b was identical to that of mellein. However, there was much less homology between the fragmentation patterns of the ochratoxins and mellein. Notably in ochratoxin b, absence of a B2/E relationship between the fragment ion m/z 204 and the molecular ion implies that the loss of water was a thermal effect. In ochratoxin a two distinct ions of m/z 194 were recognised, arising via different fragmentations of the molecular ion, the minor route being via the base peak (m/z 212). There are also two forms of the corresponding fragment ion (m/z 160), with different elemental composition, in ochratoxin b. All major fragmentation losses of mellein occur in ochratoxins a and/or b. Composition of the prominent fragment ion m/z 134 in mellein is the same as that in ochratoxin b, but there its origin was not clear. 3.7. Analytical The preparative HPLC system used a C18 column (30 2.5 cm), detection by fluorescence (excitation 340 nm, emission 400 nm) and UV absorbance (332 nm) in sequence, isocratic mobile phase H2O:CH3COOH:acetonitrile, 59.5:1:39.5 (v:v:v), at 5.6 ml min1 (Rt ochratoxins A and B, 20 and 15 min, respectively). Ochratoxins A and B were purified further in MeOH:H2O, 7:3 (v:v), Rt 21 and 16 min, respectively. Analytical HPLC of 20 ml samples in MeOH used a C18 column (15 0.5 cm), a diode array detector and
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solvent flow at 1 ml min1: Rt ochratoxins A and B, 7.8 and 4.5 min, respectively. Neutral metabolites (mellein, diaporthin and orthosporin), monitored at 239 nm, had Rt of 4.5, 3.5 and 1.8 min, respectively. TLC of portions of acidic and neutral extracts from radiolabelling experiments was performed on 10 10 cm TLC plates (SIL G, UV254 Camlab, Cambridge) in toluene–EtOAc–HCOOH, 5:4:1 (v:v:v). Dried plates were autoradiographed at 70 C for 1 week. Ochratoxins were recognised by fluorescence. Mellein was visualised by spraying with 3% FeCl3 in butan-1-ol whereby mellein is stained red (van der Merwe et al., 1965b). 3.7.1. Assay of ergosterol One gram sample of shredded wheat culture was refluxed in MeOH (100 ml) for 2 h. 4% KOH in 95% aqueous EtOH (20 ml) was added and neutral lipids were partitioned into pentane (340 ml). Solutes were dissolved in MeOH for analytical HPLC in 95% aqueous MeOH, detection at 282 nm, Rt 8 min (after Newell et al., 1988). 3.8. Treatment with ethionine Ethionine (10 ml of a 5 mg ml1 sterile solution) was added to potato dextrose broth cultures 32, 50, 56 or 72 h post-inoculation. Sterile water (10 ml) was added to a control culture. After 80 h, ochratoxins were extracted from the broth and quantified. 3.9. Relative dynamics of secondary metabolite production in submerged and solid substrate fermentations At intervals, 10 ml samples were taken from triplicate liquid cultures. Broth was filtered through a weighed sintered 10 ml glass syringe and the retained biomass
Scheme 1. EIMS fragmentation of ochratoxins a and b and mellein deduced from mass measurements and linked scan experiments.
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was lyophilised to constant weight. Acidified filtrate was extracted with EtOAc which was partitioned against bicarbonate. Ensuing acidic or neutral solutes were dissolved in MeOH for HPLC quantitation. Similarly, l g solid culture was taken at intervals and acidic and neutral metabolites analysed. 3.10. Experiments with radiolabelled precursors on isolate D2306 [1-14C]acetate (5 mCi) was added to a liquid fermentation at 8, 22.5, 32 or 50 h post inoculation to determine the time of optimal incorporation into OTA, as assessed at 80 h. A portion (8–25%) of extracts was analysed by HPLC and radioactivity in eluates corresponding to specific quantified metabolites was measured by scintillation counting. 2 g aliquots of fermenting shredded wheat were transferred to groups of three 50 ml flasks from standard flask fermentations after 3, 4, 5 and 6 days. To each was added 200 ml of sterile water containing 5 mCi of either [1-14C]acetate, l-[U-14C]phenylalanine or l-[methyl-14C]methionine. Each solution was applied to the substrate as very small drops. Incubation was continued for a further 5 h before extraction of ochratoxins to compare efficiencies of incorporation of precursors. Similarly, three 2 g amounts of 4 day culture were given 200 ml sterile water containing 0.1 mCi of either [14C]ochratoxin a, [14C]ochratoxin b or [14C]mellein and incubated for 5 h before extraction to measure fates of the radiolabeled probes. Similar fermentation material, though at the 5 day stage, was treated with dual-labeled OTA (231 mg) or OTB (1.02 mg) to explore their fate during 5 h. Again, dual-labeled OTA (1.28 mg in 3% NaHCO3) was given to 6 day culture and incubated for longer (overnight).
Acknowledgements We thank J. Barton, Chemistry Department, Imperial College for obtaining EIMS data (Micromass Autospec Q). J.P.H. acknowledges a Research Studentship from the AFRC. References Abell, C., Garson, M.J., Leeper, F.J., Staunton, J., 1982. Biosynthesis of the fungal metabolites alternariol, mellein, rubrofusarin, and 6methylsalicylic acid from CD3CO2H. Journal of the Chemical Society, Chemical Communications, 1011–1013. Abell, C., Doddrell, D.M., Garson, M.J., Laue, E.D., Staunton, J., 1983. Biosynthesis of the polyketide mellein from CD3CO2H and 13 CD3CO2H in Aspergillus melleus: detection of deuterium by 2H N.M.R. and edited 13C N.M.R. spectra. Journal of the Chemical Society Chemical Communications, 694–696. Connole, M.D., Blaney, B.J., McEwan, T., 1981. Mycotoxins in ani-
mal feeds and toxic fungi in Queensland 1971–1980. Australian Veterinary Journal 57, 314–318. de Jesus, A.E., Steyn, P.S., Vleggaar, R., Wessels, P.L., 1980. Carbon13 nuclear magnetic resonance assignments and biosynthesis of the mycotoxin ochratoxin A. Journal of the Chemical Society, Perkin Transactions 1, 52–54. Delgadillo, I., 1986. Isolation of secondary metabolites of Aspergillus ochraceus by HPLC. Mycotoxin Research 2, 9–17. Ferreira, N.P., Pitout, M.J., 1969. The biosynthesis of ochratoxin. Journal of the South African Chemical Institute 22, S1. Harris, J.P., Mantle, P.G., 2001. Biosynthesis of diaporthin and orthosporin by Aspergillus ochraceus. Phytochemistry 57, 165–169. Hesseltine, C.W., 1972. Solid state fermentations. Biotechnology and Bioengineering 14, 517–532. Huff, W.E., Hamilton, P.B., 1979. Mycotoxins—their biosynthesis in fungi: ochratoxins—metabolites of combined pathways. Journal of Food Protection 42, 815–820. Lillehoj, E.B., Aalund, O., Hald, B., 1978. Bioproduction of [14C] ochratoxin A in submerged culture. Applied and Environmental Microbiology 36, 720–723. Lindenfelser, L.A., Ciegler, A., 1975. Solid-substrate fermentor for ochratoxin A production. Applied Microbiology 29, 323–327. Mantle, P.G., McHugh, K.M., 1993. Nephrotoxic fungi in foods from nephropathy households in Bulgaria. Mycological Research 97, 205–212. Moore, J.H., Davis, N.D., Diener, U.L., 1972. Mellein and 4-hydroxymellein production by Aspergillus ochraceus Wilhelm. Applied Microbiology 23, 1067–1072. Newell, S.Y., Arsuffi, T.L., Fallon, R.D., 1988. Fundamental procedures for determining ergosterol content of decaying plant material by liquid chromatography. Applied and Environmental Microbiology 54, 1876–1879. Searcy, J.W., Davis, N.D., Diener, U.L., 1969. Biosynthesis of ochratoxin A. Applied Microbiology 18, 622–627. Shotwell, O.L., Hesseltine, C.W., Stubblefield, R.D., Sorenson, W.G., 1966. Applied Microbiology 14, 425. Stander, M.A., Steyn, P.S., Lubben, A., Miljkovic, A., Mantle, P.G., Marais, G.J., 2000. Influence of halogen salts on the production of the ochratoxins by Aspergillus ochraceus Wilh. Journal of Agricultural and Food Chemistry 48, 1865–1871. Steyn, P.S., Holzapfel, C.W., 1967. The isolation of the methyl and ethyl esters of ochratoxins A and B, metabolites of Aspergillus ochraceus Wilh. Journal of the South African Chemical Institute 20, 186–189. Steyn, P.S., Holzapfel, C.W., Ferreira, N.P., 1970. The biosynthesis of ochratoxins, metabolites of Aspergillus ochraceus. Phytochemistry 9, 1977–1983. Stoev, S.D., Goundasheva, D., Mirtcheva, T., Mantle, P.G., 2000. Susceptibility to secondary bacterial infections in growing pigs as an early response in ochratoxicosis. Experimental and Toxicologic Pathology 52, 287–296. Tapia, M.O., Seawright, A.A., 1984. Experimental ochratoxicosis A in pigs. Australian Veterinary Journal 61, 219–222. van der Merwe, K.J., Steyn, P.S., Fourie, L., de Scott, B., Theron, J.J., 1965a. Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh. Nature 205, 1112–1113. van der Merwe, K.J., Steyn, P.S., Fourie, L., 1965b. Mycotoxins. Part 2. The constitution of ochratoxin A, B, and C, metabolites of Aspergillus ochraceus Wilh. Journal of the Chemical Society, 7083–7088. Whalley, A.J.S., Edwards, R.L., 1985. Nummulariella marginata: its conidial state, secondary metabolites and taxonomic relationships. Transactions of the British Mycological Society 85, 385–390. Wei, R.-D., Strong, F.M., Smalley, E.B., 1971. Incorporation of chlorine-36 into ochratoxin A. Applied Microbiology 22, 276– 277. Yamazaki, M., Maebayashi, Y., Miyaki, K., 1971. Biosynthesis of ochratoxin A. Tetrahedron Letters 25, 2301–2303.
Biosynthesis of ochratoxins by Aspergillus ochraceus Jonathan P. Harris, Peter G. Mantle* Biochemistry Department, Imperial College of Science, Technology and Medicine, London, SW7 2AY, UK Received 19 January 2001; received in revised form 5 July 2001
Abstract Shaken liquid fermentation of an isolate of Aspergillus ochraceus showed growth-associated production of ochratoxins A and B, followed by production of a related polyketide diaporthin. Later, between 150 and 250 h, mellein accumulated transitorily. In contrast, shaken solid substrate (shredded wheat) fermentation over 14 days produced mainly ochratoxins A and B (ratio ca. 5:1) in very high yield (up to 10 mg/g). In these systems experiments with 14C-labelled precursors and putative intermediates revealed temporal separation of early and late stages of the ochratoxin biosynthetic pathway, but did not support an intermediary role for mellein. The pentaketide intermediate ochratoxin b was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin a was only biotransformed significantly (4.85%) into ochratoxin A, indicating that chlorination is mainly a penultimate biosynthetic step in the biosynthesis of ochratoxin A. This was supported by poor (1.5%) conversion of radiolabelled ochratoxin B into ochratoxin A. Experiments implied that some ochratoxin B may arise by dechlorination of ochratoxin A. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus ochraceus; Pulse-radiolabeling; Shaken solid substrate fermentation; Ochratoxin A; Ochratoxin B; Ochratoxin a; Ochratoxin b; Mellein; Ethionine
1. Introduction The mycotoxin ochratoxin A (OTA, 1) (Fig. 1), first obtained from a South African Aspergillus ochraceus isolate (van der Merwe et al., 1965a), consists of a dihydroisocoumarin moiety (the pentaketide-derived ochratoxin a, 3; Fig. 2) linked through the carboxyl group to phenylalanine. Corresponding des-chloro analogues are ochratoxins B (OTB, 2) and b (4). Unlike ochratoxin a, ochratoxin b and mellein (5) have both been isolated from ochratoxinogenic A. ochraceus fermentations (Moore et al., 1972; Delgadillo, 1986). 14 C-labelled precursor feeding experiments and subsequent chemical degradation showed that phenylalanine was incorporated into OTA, whereas ochratoxin a was constructed from five acetate units with a one carbon addition at C-7 from methionine (Ferriera and Pitout, 1969; Searcy et al., 1969; Steyn et al., 1970). A crude cell-free enzyme preparation (OTA synthetase, requiring ATP and Mg++ ions) catalysed the linking of * Corresponding author. Tel.: +44-207-594-5245; fAx: +44-207225-0960. E-mail address: [email protected] (P.G. Mantle).
ochratoxin a with phenylalanine (Ferriera and Pitout, 1969). Wei et al. (1971) demonstrated incorporation of 36 Cl into OTA. The methylation inhibitor ethionine completely inhibited OTA production (Yamazaki et al. 1971). The 13C NMR spectrum of OTA was fully assigned by de Jesus et al. (1980); ochratoxinogenic fungal cultures fed [1-13C] or [1, 2-13C2]acetate showed enhanced signals and 13C–13C coupling evidence supporting a pentaketide folding pattern similar to that in the biosynthesis of citrinin and mellein. Huff and Hamilton (1979), recognising the lack of research in the pathway of OTA biosynthesis, proposed a scheme, although this unfortunately ignored the ubiquitous OTB. Subsequently, the ochratoxin polyketide synthase was reported to produce mellein; Abell et al., (1982) showed that there was no keto-enol tautomerism, but an unfunctionalised double-bond between C-6 and C-7 in the mellein pentaketide chain. Also, the carbonyl at C-3 may have been reduced to a hydroxyl prior to folding (Abell et al., 1983). The requisite methylation step was thought to occur after the production of mellein although the subsequent putative intermediates 7methylmellein, 7-methoxymellein and 7-formylmellein
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00316-8
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limit of 5 mg/kg (5 ppb) in commodities for human food. It is timely, therefore, to contribute to the understanding of some fermentation dynamics of A. ochraceus, as one of the fungi involved in commodity spoilage, and of some later steps in the biosynthesis of ochratoxins.
2. Results and discussion 2.1. Experiments in shaken liquid fermentation
Fig. 1. Structures of the Aspergillus ochraceus mycotoxins ochratoxins A and B.
Fig. 2. Structures of the dihydroisocoumarins ochratoxins a and b and mellein.
have not been isolated from fungal fermentation producing ochratoxins. Chlorination of ochratoxin b by a chloroperoxidase was reasoned as occurring prior to joining the aromatic nucleus to phenylalanine via an acyl activated phosphoochratoxin a reacting with phenylalanine with its carboxy group protected by ethyl esterification. This reaction would result in ochratoxin C which could be transesterified to the OTA methyl ester, both of which OTA derivatives have been isolated from A. ochraceus cultures (van der Merwe et al. 1965b; Steyn and Holzapfel, 1967). Alternatively, ochratoxin C might be hydrolysed directly to OTA. OTA, as a nephrotoxin and a carcinogen, is currently the subject of proposed EC legislation to place an upper
2.1.1. Culture dynamics In potato dextrose broth, accumulation of biomass was complete within 3–4 days and was closely associated with production of ochratoxins (Fig. 3) (also noted by Steyn et al., 1970; Wei et al., 1971). There were also second and third temporal phases of secondary metabolite occurrence measured concerning the polyketides diaporthin (6; Fig. 4) and mellein. Throughout the phase of ochratoxin occurrence there was a ratio of A:B of approximately 3:1. Diaporthin was also accompanied by approximately half the amount of orthosporin (de-O-methyl diaporthin; Harris and Mantle, 2001). The transitory appearance of mellein might plausibly be interpreted as reflecting cessation of involvement of this putative intermediate in ochratoxin biosynthesis after growth-associated production of the phenylalanine moiety declined. Alternatively, the observed temporal sequence of metabolites simply reflected progressive decline in biosynthetic complexity from ochratoxins (requiring phenylalanine, methionine, acetate and chlorine) to mellein which is solely derived from acetate. 2.1.2. Effect of ethionine The only marked effect of the methylation inhibitor ethionine on accumulation of ochratoxins in submerged fermentation was on yield of OTA when added during early idiophase at 32 h (Table 1). This is consistent with methylation of the pentaketide occurring early in biosynthesis. The less obvious or nil effect on excreted OTB would be consistent with some of this arising by dechlorination of OTA. 2.1.3. Radioactivity experiment Radiolabel from [1-14C]acetate was incorporated into OTA when added as early as only 8 h into the fermentation; the finding demonstrated the close association of ochratoxin biosynthesis with replicatory growth (Table 2). The optimum time of addition of the polyketide precursor to achieve maximum specific radioactivity in OTA was at 22.5 h, consistent with a principle of maximum rate of isocoumarin biosynthesis preceding prominent occurrence of OTA in broth (Fig. 3). Reinterpretation of the data of Lillehoj et al.
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Fig. 3. Dry cell weight (DCW), diaporthin ([Diap]), ochratoxin A ([OTA]), ochratoxin B ([OTB]) and mellein ([Mellein]) concentration versus time for an A. ochraceus fermentation in potato dextrose broth. Table 2 Effect of adding [1-14C]acetate (5 mCi) to potato dextrose broth fermentations at different stages on the amount and specific radioactivity of [14C]ochratoxin A recovered and analysed 80 h after inoculation
Fig. 4. Structure of diaporthin (6).
Table 1 Effect of adding ethionine (50 mg) at different fermentation stages in potato dextrose broth on the concentrations of ochratoxins A and B measured 80 h after inoculation
Addition time (h)
Ochratoxin A (mg)
Specific radioactivity (dpm/mg)
8 22.5 32 50
23 143 143 152
6 37 18 15
2.2. Experiments in shaken solid substrate fermentation
ochraceus growth in shaken shredded wheat substrate (Fig. 5). Accumulation of OTA and OTB closely followed fungal growth and was near maximum after 2 weeks; a 5:1 ratio up to day 9 diverged later in favour of OTB, possibly reflecting limitation of substrate chlorine ions for OTA formation (Stander et al., 2000). Yields of 10 mg OTA/g substrate have been achieved. If ergosterol were even as much as 1% of A. ochraceus mycelial biomass, OTA would become 1/10th of fungal biomass; this is probably a conservative estimate. Mellein, diaporthin and orthosporin were absent or barely detected throughout this two-week fermentation. Contentious aspects of ochratoxin biosynthesis were therefore explored in this unusual medium in which secondary metabolism in A. ochraceus was highly focused on the ochratoxin pathway.
2.2.1. Culture dynamics Ergosterol, as a measure of fungal mycelium in the substrate, increased linearly through days 4–8 of A.
2.2.2. Radioactivity experiments The magnitude of the relative incorporations of the three radioactive biosynthetic precursors into OTA and
Ethionine addition times (h)
[Ochratoxin A] (mg/l)
[Ochratoxin B] (mg/l)
32 50 56 72 Control
2.1 4.2 3.7 4.5 4.8
1.2 1.3 1.3 1.3 1.4
(1978) reveals evidence of a similar temporal separation of steps in ochratoxin biosynthesis.
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Fig. 5. Changes in ergosterol ([Ergo]), ochratoxin A ([OTA]) and ocratoxin B ([OTB]) concentraion during 2 weeks of an Aspergillus ochraceus solid substrate fermentation.
OTB, when added to fermentations on days 3, 4, 5 or 6 (Figs. 6 and 7), showed that the experimental system was well suited to biosynthetic study. The data also demonstrated temporal separation of optima for isocoumarin biosynthesis (day 4 for OTA; days 4–5 for OTB) and addition of the phenylalanine moiety (day 5 for OTA; days 5–6 for OTB). Notably, 50% of the l[U-14C]phenylalanine added at 5 days was incorporated into OTA and OTB. Relative incorporations of the putative intermediates ochratoxins a and b and mellein (Table 3) indicated a strong preferential role of a into A, but similar strong involvement of b into both A and B as subsequently isolated. 14C from mellein occurred in much smaller amount in ochratoxins, which may only reflect indirect incorporation of the radiolabel from mellein catabolism. To suggest that the rather negative experimental result arose from poor uptake of added mellein seems weak when added ochratoxins a and b, both only having an additional carboxyl group, were incorporated so extensively into OTA and OTB. Mellein was only a transitory product in shaken liquid culture (Fig. 3) and therefore A. ochraceus is well able to degrade it; no trace of mellein was found in the fermentation fed [14C]mellein. Indeed, the same putative indirect incorporation may explain the small amount of radiolabel in OTB after feeding with ochratoxin a. Autoradiography of chromatograms of neutral and acidic components of extracts of cultures fed ochratoxin a showed some 14C in neutral non-polar components, in
Fig. 6. The % incorporation of 5 mCi of either [1-14C]acetate, [methyl-14C]methionine or [U-14C]phenylalanine into [14C]ochratoxin A during 5 h after adding to Aspergillus ochraceus solid substrate fermentations on days 3, 4, 5 and 6.
addition to qualitative confirmation of incorporation into ochratoxins. This supports the interpretation of only indirect incorporation from ochratoxin a to OTB. Conversely, there was no autoradiographic evidence of biotransformations in the fermentation given ochratoxin b other than into OTA and OTB. In the fermentation given [14C]mellein there were no particular locations of radiolabel in any fraction of extract, supporting the notion of its rather rapid and extensive catabolism. In following the fate of dual-labeled OTA and OTB in shaken shredded wheat culture, more than 40% of the added OTA was recovered in the form as added (Table 4). However, an amount equivalent to ca.10% was present as OTB although with the 3H:14C ratio being slightly different. If this is a real change it might imply that the phenylalanine moiety can be transferred to OTB more readily than can the dihydroisocoumarin moiety, which would have to be dechlorinated. Very little (approx. 6.5%) of the added dual-labeled OTB was recovered as such with a similar 3H:14C ratio and even less was incorporated into OTA. Thus, experimental change of doublylabelled OTA into OTB 5 h after it was added to a fermentation in early idiophase (Table 4) was the most significant interconversion observed. Generally maintained
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Fig. 7. The % incorporation of 5 mCi of either [1-14C]acetate, [methyl-14C]methionine or [U-14C]phenylalanine into [14C]ochratoxin B during 5 h after adding to Aspergillus ochraceus solid substrate fermentations on days 3, 4, 5 and 6.
Table 3 The % incorporation of 0.13 mCi [14C]ochratoxin a, 0.1 mCi [14C] ochratoxin b or 0.096 mCi [14C]mellein into [14C]ochratoxins A and B during 5 h of solid substrate fermentation at the 4 day stage
Ochratoxin A Ochratoxin B
3
[14C]ochratoxin a
[14C]ochratoxin b
[14C]mellein
4.85 0.47
14.32 18.98
0.56 0.68
H:14C ratios confirmed the validity of most of the transformations observed. Correct interpretation of the findings depends on the extent of cellular uptake of the compounds administered; conversion of OTA to OTB as a catabolic step could be extracellular, but chlorination of OTB to OTA probably requires cellular uptake. If there is not an uptake mechanism for OTB, this could impose a limit on experimentation. It is concluded from all the experiments that chlorination of both ochratoxin b and OTB can give rise to OTA. However, there was no evidence for the intermediate role of the ester ochratoxin C proposed by Huff and Hamilton (1979), presumably interpreted as having a role in protecting the phenylalanine carboxyl during the last biosynthetic steps. These results suggest that one, possibly dominant,
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biosynthetic pathway involves ochratoxins b!a!A, with also a branch for ochratoxins b!B. An alternative pathway might be ochratoxins b!B!A but, if ochratoxin a is not directly involved, it is difficult to explain the present demonstration that it can occur naturally. However, mellein seems not to be involved as a precursor of either pathway. The discovery of 5-methyl-, 5formyl- and 5-carboxymellein in culture broth of Nummulariella marginata (Whalley and Edwards, 1985) provides a precedent for an oxidation system for a substituted methyl group, yet if such compounds are stable enough to be isolated from fermentation broth it is questioned why 7-methyl- and 7-formylmellein have not, so far, been discovered. Results of the second duallabeling experiment complemented the first (Table 4). Over half the added OTA was recovered and only 3% was incorporated into OTB although with a slightly different radionuclide ratio. Autoradiography of culture extract chromatograms showed no 14C in neutral fractions, in contrast to results from experiments with [14C] ochratoxins a and b and mellein. Where [14C] OTA or OTB was added, radioactivity was only obvious in the unchanged ochratoxin corresponding to what had been added. Thus, although it was shown that OTA can be converted into OTB nonspecifically, there was no convincing evidence whether or not specific biotransformation of OTB into OTA had occurred. Since the dual-labelled experiments did not achieve the high percentage incorporations of the experiments in which ochratoxins a and b were administered, it could be argued that the inter-conversion of OTA and OTB, although shown to occur, is not as important in the biosynthetic pathway as the reactions involving ochratoxins a and b.
3. Experimental 3.1. Fungi Two isolates of A. ochraceus were used; D2306 for its high yield of ochratoxins (Connole et al. 1981; as also used since by Tapia and Seawright, 1984, Stander et al., 2000; Stoev et al., 2000) and KBf for its production of mellein (though not producing ochratoxins; Mantle and McHugh, 1993). The fungi were maintained on potato dextrose agar (Difco). 3.2. Culture conditions Submerged liquid culture was in potato dextrose broth (Difco), 100 ml in 500 ml Erlenmeyer flasks, inoculated with spores to give 2–4106 spores ml1 and incubated on an orbital shaker (200 rpm, 10 cm eccentric throw), at 28 C for isolate D2306 and 22 C for isolate KBf.
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Table 4 The % incorporation and 3H:14C ratio of ochratoxins A and B recovered after adding dual-labelled ochratoxins A and B to solid substrate fermentations % Incorporation
3
Ochratoxin A added for 5 h on day 5 3 H Ochratoxin A 14 C 3 Ochratoxin B H 14 C
43.2 41.5 12.1 8.2
1:0.47
Ochratoxin B added for 5 h on day 5 3 H Ohratoxin A 14 C 3 Ochratoxin B H 14 C
1.4 1.5 6.7 6.4
1:0.43
Experiment
Isotope
Ochratoxin A added on day 6 and incubated overnight 3 H 58.3 Ochratoxin A 14 C 57.5 3 Ochratoxin B H 3.3 14 C 2
H:14C
on day 4, were each hydrolysed in 50 ml 6 M HCl by reflux for 18 h. The cooled mixture was extracted with EtOAc and ochratoxins a and b were purified by HPLC (see below) detected at 323 nm (retention time, Rt, 10 and 6 min, respectively, at 8.4 ml min1). 3.5. Biosynthetic preparation of [14C]mellein
1:0.48
Sodium [1-14C]acetate (10 mCi) was added to a 37 h liquid fermentation flask culture of isolate KBf and the fermentation continued for 1 h. [14C] Mellein (2.22 mg) was obtained by extraction of culture filtrate with EtOAc followed by C18 SepPak and HPLC purification (see below). The measured radioactivity (1.67 mCi) represented 16.7% incorporation of radiolabel from [1-14C]acetate.
1:0.29
3.6. Source of authentic compounds
1:0.33
1:0.37
Shaken solid substrate fermentation in Erlenmeyer flasks was developed from the principle first devised for aflatoxins (Shotwell et al., 1966), then applied to OTA (Hesseltine, 1972) and scaled up to rotating barrel fermenters (Lindenfelser and Ciegler, 1975). Shredded wheat breakfast cereal (Cereal Partners UK, Welwyn Garden City), 40 g per flask, was autoclaved, moistened to an initial moisture content of approximately 35% with sterile water (16 ml) and incubated on the orbital shaker at 28 C. Sterile water was also added at 24 and 48 h (4 and 2 ml, respectively). Water evaporation and metabolic dissimilation of substrate during the subsequent 2 weeks of shaken fermentation resulted in approximately 35 g of moist chocolate-coloured granules in each flask. Some small-scale fermentations were performed as 2 g of shredded wheat in 50 ml flasks for radiochemical experiments. 3.3. Biosynthetic preparation of dual-labeled OTA and OTB After 4 days in standard shredded wheat culture, 2 g material was transferred to a 50 ml flask and given 5 mCi each of [methyl-14C]methionine and l-[2, 3, 4, 5, 6-3H]phenylalanine. Shaken incubation continued for 5 h and the culture was extracted for OTA (3.4 mg, 0.15 mCi 3H:0.07 mCi 14C, ratio 3H:14C 1: 0.47) and OTB (1.5 mg, 0.29 mCi 3H:0.11 mCi 14C, ratio 3H:14C 1: 0.38). Radioactivity from phenylalanine was incorporated about twice as efficiently as that from methionine. 3.4. Preparation of 14C labeled ochratoxins and OTA (1.16 mg, 0.21 mCi) and OTB (1.44 mg, 0.14 mCi), from cultures fed [methyl-14C]methionine for 5 h
OTA and OTB were isolated from solid substrates (2 g) by extraction in 100 ml EtOAc:0.01 M H3PO4, 9:1 (v:v), partition into aqueous NaHCO3 (3%), acidified and back extracted into EtOAc. Residues were taken up in MeOH. OTA and OTB were purified by preparative HPLC (see later). Ochratoxins a and b were synthesized by the acid hydrolysis of 1 mg OTA or OTB, respectively, followed by preparative HPLC (see later). Mellein was obtained from 1 week potato dextrose broth culture of isolate KBf. The filtered broth, adjusted to pH 3, was extracted with EtOAc (100 ml) and the organic phase partitioned against a 3% aqueous NaHCO3 solution to remove acidic metabolites. The organic extract was taken to dryness. The solutes, dissolved in MeOH, were processed by HPLC (below; detection at 239 nm, Rt 30 min). Combined eluates corresponding to mellein were mixed with an equal volume of H2O and passed through a C18 SepPak classic cartridge pre-conditioned first with MeOH (20 ml) and then with H2O (20 ml). Mellein was eluted in EtOH in the 2nd and 3rd 0.5 ml fraction. Diaporthin and orthosporin were available from other studies (Harris and Mantle, 2001). All compounds were investigated by HR-MS, and linked scan EIMS (B/E and B2/E) to interpret fragmentation. The EI mass spectra of OTA and OTB differ by the chlorine or proton substituent at C5, respectively. Molecular ions are very weak, as are those corresponding to losses of H2O or CO2. Therefore, use of EIMS for confirming OTA relies on the two important fragment ions, in this case m/z 255 and 239 (base peak). These represent, respectively, the isocoumarin moiety still with the amino nitrogen of phenylalanine and the isocoumarin alone. Principles of further EI fragmentation of the latter, with a carboxyl group reconstituted at C10, are illustrated by ochratoxin a. It is helpful to have a rationale for occurrence of fragment ions in EIMS at
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least for diagnostic purposes and this has not been available before. From accurate mass measurements and linked-scan analysis of ochratoxins a and b, and of mellein, rationalised MS fragmentation [Scheme 1, extending the data of Delgadillo (1986)] showed considerable homology between ochratoxins a and b. Also, the composition of ion m/z 149 in ochratoxin b was identical to that of mellein. However, there was much less homology between the fragmentation patterns of the ochratoxins and mellein. Notably in ochratoxin b, absence of a B2/E relationship between the fragment ion m/z 204 and the molecular ion implies that the loss of water was a thermal effect. In ochratoxin a two distinct ions of m/z 194 were recognised, arising via different fragmentations of the molecular ion, the minor route being via the base peak (m/z 212). There are also two forms of the corresponding fragment ion (m/z 160), with different elemental composition, in ochratoxin b. All major fragmentation losses of mellein occur in ochratoxins a and/or b. Composition of the prominent fragment ion m/z 134 in mellein is the same as that in ochratoxin b, but there its origin was not clear. 3.7. Analytical The preparative HPLC system used a C18 column (30 2.5 cm), detection by fluorescence (excitation 340 nm, emission 400 nm) and UV absorbance (332 nm) in sequence, isocratic mobile phase H2O:CH3COOH:acetonitrile, 59.5:1:39.5 (v:v:v), at 5.6 ml min1 (Rt ochratoxins A and B, 20 and 15 min, respectively). Ochratoxins A and B were purified further in MeOH:H2O, 7:3 (v:v), Rt 21 and 16 min, respectively. Analytical HPLC of 20 ml samples in MeOH used a C18 column (15 0.5 cm), a diode array detector and
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solvent flow at 1 ml min1: Rt ochratoxins A and B, 7.8 and 4.5 min, respectively. Neutral metabolites (mellein, diaporthin and orthosporin), monitored at 239 nm, had Rt of 4.5, 3.5 and 1.8 min, respectively. TLC of portions of acidic and neutral extracts from radiolabelling experiments was performed on 10 10 cm TLC plates (SIL G, UV254 Camlab, Cambridge) in toluene–EtOAc–HCOOH, 5:4:1 (v:v:v). Dried plates were autoradiographed at 70 C for 1 week. Ochratoxins were recognised by fluorescence. Mellein was visualised by spraying with 3% FeCl3 in butan-1-ol whereby mellein is stained red (van der Merwe et al., 1965b). 3.7.1. Assay of ergosterol One gram sample of shredded wheat culture was refluxed in MeOH (100 ml) for 2 h. 4% KOH in 95% aqueous EtOH (20 ml) was added and neutral lipids were partitioned into pentane (340 ml). Solutes were dissolved in MeOH for analytical HPLC in 95% aqueous MeOH, detection at 282 nm, Rt 8 min (after Newell et al., 1988). 3.8. Treatment with ethionine Ethionine (10 ml of a 5 mg ml1 sterile solution) was added to potato dextrose broth cultures 32, 50, 56 or 72 h post-inoculation. Sterile water (10 ml) was added to a control culture. After 80 h, ochratoxins were extracted from the broth and quantified. 3.9. Relative dynamics of secondary metabolite production in submerged and solid substrate fermentations At intervals, 10 ml samples were taken from triplicate liquid cultures. Broth was filtered through a weighed sintered 10 ml glass syringe and the retained biomass
Scheme 1. EIMS fragmentation of ochratoxins a and b and mellein deduced from mass measurements and linked scan experiments.
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was lyophilised to constant weight. Acidified filtrate was extracted with EtOAc which was partitioned against bicarbonate. Ensuing acidic or neutral solutes were dissolved in MeOH for HPLC quantitation. Similarly, l g solid culture was taken at intervals and acidic and neutral metabolites analysed. 3.10. Experiments with radiolabelled precursors on isolate D2306 [1-14C]acetate (5 mCi) was added to a liquid fermentation at 8, 22.5, 32 or 50 h post inoculation to determine the time of optimal incorporation into OTA, as assessed at 80 h. A portion (8–25%) of extracts was analysed by HPLC and radioactivity in eluates corresponding to specific quantified metabolites was measured by scintillation counting. 2 g aliquots of fermenting shredded wheat were transferred to groups of three 50 ml flasks from standard flask fermentations after 3, 4, 5 and 6 days. To each was added 200 ml of sterile water containing 5 mCi of either [1-14C]acetate, l-[U-14C]phenylalanine or l-[methyl-14C]methionine. Each solution was applied to the substrate as very small drops. Incubation was continued for a further 5 h before extraction of ochratoxins to compare efficiencies of incorporation of precursors. Similarly, three 2 g amounts of 4 day culture were given 200 ml sterile water containing 0.1 mCi of either [14C]ochratoxin a, [14C]ochratoxin b or [14C]mellein and incubated for 5 h before extraction to measure fates of the radiolabeled probes. Similar fermentation material, though at the 5 day stage, was treated with dual-labeled OTA (231 mg) or OTB (1.02 mg) to explore their fate during 5 h. Again, dual-labeled OTA (1.28 mg in 3% NaHCO3) was given to 6 day culture and incubated for longer (overnight).
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