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Applied and Environmental Microbiology, August 2001, p. 3514-3522, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3514-3522.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The P450-4 Gene of Gibberella fujikuroi Encodes ent-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway

Bettina Tudzynski,1,* Peter Hedden,2 Esther Carrera,2 and Paul Gaskin2

Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schloßgarten 3, D-48149 Münster, Germany,1 and IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom2

Received 28 February 2001/Accepted 23 May 2001

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA4. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3' consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.

* Corresponding author. Mailing address: Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schloßgarten 3, D-48149 Münster, Germany. Phone: (49) 251.832-24801. Fax: (49)251.8323823. E-mail: Bettina.Tudzynski{at}uni-muenster.de.

Applied and Environmental Microbiology, August 2001, p. 3514-3522, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3514-3522.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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