Elsevier

Phytochemistry

Volume 58, Issue 5, November 2001, Pages 709-716
Phytochemistry

Biosynthesis of ochratoxins by Aspergillus ochraceus

https://doi.org/10.1016/S0031-9422(01)00316-8Get rights and content

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 β was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin α 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.

Radiochemical experiments in shaken solid substrate fermentation of Aspergillus ochraceus Wilhelm showed efficient incorporation of ochratoxin β into both ochratoxins A and B; the chlorinated analogue ochratoxin α was only a significant precursor of ochratoxin A, indicating chlorination as mainly a penultimate biosynthetic step. The fungus was able to produce the polyketide mellein but experimental data did not support its putative role as a precursor of ochratoxins.

  1. Download : Download full-size image

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 α, 3; Fig. 2) linked through the carboxyl group to phenylalanine. Corresponding des-chloro analogues are ochratoxins B (OTB, 2) and β (4). Unlike ochratoxin α, ochratoxin β and mellein (5) have both been isolated from ochratoxinogenic A. ochraceus fermentations (Moore et al., 1972, Delgadillo, 1986).

14C-labelled precursor feeding experiments and subsequent chemical degradation showed that phenylalanine was incorporated into OTA, whereas ochratoxin α was constructed from five acetate units with a one carbon addition at C-7 from methionine (Ferreira & 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 ochratoxin α with phenylalanine (Ferriera and Pitout, 1969). Wei et al. (1971) demonstrated incorporation of 36Cl 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 7-methylmellein, 7-methoxymellein and 7-formylmellein have not been isolated from fungal fermentation producing ochratoxins.

Chlorination of ochratoxin β by a chloroperoxidase was reasoned as occurring prior to joining the aromatic nucleus to phenylalanine via an acyl activated phospho-ochratoxin α reacting with phenylalanine with its carboxy group protected by ethyl esterification. This reaction would result in ochratoxin C which could be trans-esterified to the OTA methyl ester, both of which OTA derivatives have been isolated from A. ochraceus cultures (van der Merwe et al., 1965b, Steyn & 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 limit of 5 μg/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.

Section snippets

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

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 & 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).

Culture conditions

Submerged liquid culture was in potato dextrose broth (Difco), 100 ml in 500 ml Erlenmeyer flasks, inoculated with spores to give 2–4×106 spores ml−1

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 (26)

  • I. Delgadillo

    Isolation of secondary metabolites of Aspergillus ochraceus by HPLC

    Mycotoxin Research

    (1986)
  • N.P. Ferreira et al.

    The biosynthesis of ochratoxin

    Journal of the South African Chemical Institute

    (1969)
  • C.W. Hesseltine

    Solid state fermentations

    Biotechnology and Bioengineering

    (1972)
  • Cited by (0)

    View full text