ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi. In plants, ent-kaurene is synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase and ent-kaurene synthase. A cell-free system of the fungus Phaeosphaeria sp. L487 converted labeled geranylgeranyl diphosphate to ent-kaurene. A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes. Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide. The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively. A fusion protein produced by expression of the cDNA in Escherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene. Amo-1618 completely inhibited the copalyl diphosphate synthase activity of the enzyme at 10 ؊6 M, whereas it did not inhibit the ent-kaurene synthase activity even at 10These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate catalytic sites for the two cyclization reactions. Gibberellins (GAs) 1 are one of an important group of phytohormones regulating many aspects of plant growth and development. Some fungal species produce GAs as secondary metabolites (1). Gibberella fujikuroi is a rice pathogenic fungus producing high amount of GAs. The fermentation and biosynthesis of GA in G. fujikuroi were well characterized, since some GAs are produced industrially using the fungus (2, 3).GA is unequivocally synthesized from ent-kaurene in both fungi and plants (4,5). ent-Kaurene is a tetracyclic diterpene hydrocarbon formed from geranylgeranyl diphosphate (GGDP) via copalyl diphosphate (CDP). The pathway of ent-kaurene biosynthesis was first confirmed using cell-free systems from G. fujikuroi (6). The two-step cyclization was thought to involve two different enzymes: copalyl diphosphate synthase (CPS, formerly ent-kaurene synthase A) and ent-kaurene synthase (KS, formerly ent-kaurene synthase B). These enzymes were partially purified from Fusarium moniliforme, an anamorph of G. fujikuroi. (7). It was suggested that CPS and KS in the fungus might be tightly associated, because the two activities could not be separated. On the other hand, the two activities were successfully separated from a plant enzyme preparation from Marah macrocarpus by chromatographic methods (8). The two enzymes are possibly localized in plastids (9). Quite recently, genes encoding both enzymes have been cloned from plants: CPS from Arabidopsis (10), maize (11) and pea (12), and KS from pumpkin (13). Several other GA biosynthetic enzymes have been cloned from plants as well (14). In contrast, no GA biosynth...