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J. Biol. Chem., Vol. 278, Issue 31, 28635-28643, August 1, 2003

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Characterization of the Final Two Genes of the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi

des AND P450-3 ENCODE GA₄ DESATURASE AND THE 13-HYDROXYLASE, RESPECTIVELY^*

Bettina Tudzynski  , Martina Mihlan , María Cecilia Rojas ¶, Pia Linnemannstöns , Paul Gaskin || and Peter Hedden ||

From the Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlogarten 3, D-48149 Münster, Germany, ^¶Laboratorio de Bioorgánica, Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile, and ^||Rothamsted Research at Long Ashton, Long Ashton, Bristol BS41 9AF, United Kingdom

Recently, six genes of the gibberellin (GA) biosynthesis gene cluster in Gibberella fujikuroi were cloned and the functions of five of these genes were determined. Here we describe the function of the sixth gene, P450–3, and the cloning and functional analysis of a seventh gene, orf3, located at the left border of the gene cluster. We have thereby defined the complete GA biosynthesis gene cluster in this fungus. The predicted amino acid sequence of orf3 revealed no close homology to known proteins. High performance liquid chromatography and gas chromatography-mass spectrometry analyses of the culture fluid of knock-out mutants identified GA₁ and GA₄, rather than GA₃ and GA₇, as the major C₁₉-GA products, suggesting that orf3 encodes the GA₄ 1,2-desaturase. This was confirmed by transformation of the SG139 mutant, which lacks the GA biosynthesis gene cluster, with the desaturase gene renamed des. The transformants converted GA₄ to GA₇, and also metabolized GA₉ (3-deoxyGA₄) to GA₁₂₀ (1,2-didehydroGA₉), but the 2-hydroxylated compound GA₄₀ was the major product in this case. We demonstrate also by gene disruption that P450-3, one of the four cytochrome P450 monooxygenase genes in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the conversion of GA₇ to GA₃, in the last step of the pathway. This enzyme also catalyzes the 13-hydroxylation of GA₄ to GA₁. Disruption of the des gene in an UV-induced P450–3 mutant produced a double mutant lacking both desaturase and 13-hydroxylase activities that accumulated high amounts of the commercially important GA₄. The des and P450–3 genes differ in their regulation by nitrogen metabolite repression. In common with the other five GA biosynthesis genes, expression of the desaturase gene is repressed by high amounts of nitrogen in the culture medium, whereas P450-3 is the only gene in the cluster not repressed by nitrogen.

Received for publication, February 24, 2003 , and in revised form, April 30, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ417493.

^* This work was supported by AGTROL (Houston, TX), the Deutsche Forschungsgemeinschaft (Tu101-7) and the Deutscher Akademischer Austausdidienst/Consejo Nacional de Ciencia y Tecnología Cooperation Program and Fondo Nacional de Desarrollo Científico y Tecnológico (Grant 1020140). Rothamsted 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.

To whom correspondence should be addressed. Tel.: 49-251-83224801; Fax: 49-251-8323823; E-mail: Bettina.Tudzynski{at}uni-muenster.de.

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