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Applied and Environmental Microbiology, June 2004, p. 3239-3245, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3239-3245.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Metabolism of Zearalenone by Genetically Modified Organisms Expressing the Detoxification Gene from Clonostachys rosea

Naoko Takahashi-Ando,1 Shuichi Ohsato,1 Takehiko Shibata,2 Hiroshi Hamamoto,3 Isamu Yamaguchi,1,3 and Makoto Kimura1,2*

Laboratory for Remediation Research, Plant Science Center,1 Cellular and Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198,2 Laboratory for Adaptation and Resistance, Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan3

Received 2 September 2003/ Accepted 23 February 2004

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45°C and pH 10.5 (kcat at 30°C, 0.51 s–1). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the kcat was highest with zearalanone (1.5 s–1) and lowest with ß-zearalenol (0.075 s–1). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, ß-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuäne, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.

* Corresponding author. Mailing address: Laboratory for Remediation Research, Plant Science Center, RIKEN, 2-1 Hirosawa, Saitama 351-0198, Japan. Phone: 81-48-467-9796. Fax: 81-48-462-4394. E-mail: mkimura{at}postman.riken.go.jp.

Applied and Environmental Microbiology, June 2004, p. 3239-3245, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3239-3245.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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  • Poppenberger, B., Berthiller, F., Bachmann, H., Lucyshyn, D., Peterbauer, C., Mitterbauer, R., Schuhmacher, R., Krska, R., Glossl, J., Adam, G. (2006). Heterologous Expression of Arabidopsis UDP-Glucosyltransferases in Saccharomyces cerevisiae for Production of Zearalenone-4-O-Glucoside.. Appl. Environ. Microbiol. 72: 4404-4410 [Abstract] [Full Text]  


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