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

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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.