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J. Biol. Chem., Vol. 279, Issue 24, 25075-25084, June 11, 2004

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The NADPH-cytochrome P450 Reductase Gene from Gibberella fujikuroi Is Essential for Gibberellin Biosynthesis^*

Stefan Malonek, Maria C. Rojas, Peter Hedden¶, Paul Gaskin¶, Paul Hopkins¶, and Bettina Tudzynski||

From the Institut für Botanik der Westfälischen Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, Germany, Laboratorio de Bioorgánica, Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile, and ^¶Rothamsted Research, Harpenden, Herts AL5 2LQ, United Kingdom

The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN, and NADPH binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA₃, GA₄, and GA₇ production, but low levels of non-hydroxylated C₂₀-GAs (GA₁₅ and GA₂₄) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs. Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.

Received for publication, August 4, 2003 , and in revised form, March 16, 2004.

^* This work was supported by the Deutsche Forschungsgemeinschaft (Tu101/9-1) and Fondo Nacional de Desarrollo Cientifico y Tecnologico Grant 1020140. Rothamstead Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ576025.

^|| To whom correspondence should be addressed. Tel.: 49-251-832-24801; Fax: 49-251-8323823; E-mail: Bettina.Tudzynski{at}uni-muenster.de.

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