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

Author:

Tudzynski Bettina1,Hedden Peter2,Carrera Esther2,Gaskin Paul2

Affiliation:

1. Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schloßgarten 3, D-48149 Münster, Germany,1 and

2. IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom2

Abstract

ABSTRACT 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 GA 4 . 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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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