Cleavage of zearalenone by Trichosporon mycotoxinivorans to a novel nonestrogenic metabolite

Abstract

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C(18)H(24)O(7), was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H](-) and at m/z 375.1416 [M+Na](+). Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6'. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay.

FIG. 1.

Comparison (large graph) of the total wavelength chromatogram (TWC) of the metabolized sample (red curve) and the culture (green curve) and the substrate (blue curve) control samples after 144 h of incubation. In the metabolized sample the substance causing the most intense peak (at 18.2 min) was identified as the ZON metabolite (ZOM-1). Depiction (inset) of estimated concentration of the ZON metabolite ZOM-1 during an incubation experiment of 10 mg/liter ZON with Trichosporon mycotoxinivorans.

FIG. 2.

(a) Full-scan spectrum (ESI negative mode) of the ZON metabolite at 18.2 min (144 h of incubation time) after background subtraction. Highlighted are the deprotonated molecular ion [M-H]⁻ at m/z 351 and further signals of its sodium and potassium adducts (m/z 373, m/z 389) as well as the molecular dimer ion at m/z 703 and further alkali-bridged dimer ions at m/z 725, 741, 747, and 763 (zoomed). (b) Structure of the ZON metabolite (ZOM-1) of T. mycotoxinivorans and consecutive numbering of the carbon atoms. Enhanced product ion data of its deprotonated molecule ion [M-H]⁻ at m/z 351 and scheme of proposed major fragmentation pathways. MW indicates molecular weight.

FIG. 3.

(A) Growth of strains YZRM7 and YZGA376 observed with SC-His-Ura medium plates after 3 days of incubation at 30°C either with ZON or with the ZON metabolite (ZOM-1) at different concentrations. Whereas a concentration of 3 nM ZON (∼1 μg/liter ZON) allows growth of the bioassay strain YZRM7 due to activation of the estrogen-inducible URA3 gene, a 1,000-fold-higher concentration of the ZON metabolite does not. (B) Detection of the luminescent product of β-galactosidase activity released after 90 min of incubation. Competitive displacement of the estrogen receptor from the estrogen-linked donor peptide by ZON reconstitutes β-galactosidase activity, while the ZON metabolite is inactive.

FIG. 4.

Description of the proposed lactone ring opening of zearalenone by Trichosporon mycotoxinivorans (a) and by Gliocladium roseum/Clonostachys rosea (b). (a) Hypothetical pathway of ZOM-1 formation. The macrocyclic ring is first extended by insertion of an oxygen next to the carbonyl group (Baeyer-Villiger oxidation), and the thereby newly formed lactone is then opened in a second step to give ZOM-1. (b) The preexisting lactone in ZON is hydrolyzed, and following decarboxylation the cleavage product 1, which can isomerize at room temperature (RT) to the cleavage product 2, is formed.