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

Abstract

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 GA 1 and GA 4 , rather than GA 3 and GA 7 , as the major C 19 -GA products, suggesting that orf3 encodes the GA 4 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 GA 4 to GA 7 , and also metabolized GA 9 (3-deoxyGA 4 ) to GA 120 (1,2-didehydroGA 9 ), but the 2␣-hydroxylated compound GA 40 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 GA 7 to GA 3 , in the last step of the pathway. This enzyme also catalyzes the 13-hydroxylation of GA 4 to GA 1 . 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 GA 4 . 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.

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

“…P450-2 converts GA 14 to GA 4 and GA 12 to GA 9 with the loss of carbon-20 (7). Finally, P450-3 is responsible for the 13-hydroxylation of GA 7 to GA 3 , and, in a minor pathway, of GA 4 to GA 1 (8). Although not all P450-catalyzed steps are completely blocked in the deletion mutants, they are reduced in activity and some reactions, such as 3␤-hydroxylation and loss of C-20, are completely absent in ⌬cpr mutants.…”

“…The major GA in most strains of G. fujikuroi is gibberellic acid (GA 3 ), the biosynthesis of which requires seven genes that are arranged in a gene cluster. Each of these genes and the function of the encoded enzymes have recently been fully characterized (3)(4)(5)(6)(7)(8). As well as genes encoding a pathway-specific geranylgeranyl diphosphate synthase (ggs2), ent-copalyldiphosphate/ent-kaurene synthase (cps/ks), and GA 4 desaturase (des), the cluster contains four cytochrome P450 monooxygenase genes (P450-1 to P450-4), which in most cases encode multifunctional enzymes that catalyze several biosynthetic steps.…”

“…The complete GA complement produced by the different strains was determined by GC-MS analysis after extraction from the culture fluid as already described (8), except that compounds were separated on a 30-m ϫ 0.32-mm ϫ 0.25-m HP-5 WCOT column (Agilent Technologies) and analyzed using a MAT95XP mass spectrometer (Thermo Electron Corp.) GC-MS conditions were as described previously (8). Compounds were identified by comparison of their mass spectra with those in a spectral library (49).…”

Gibberellin biosynthesis in bacteria: Separate ent‐copalyl diphosphate and ent‐kaurene synthases in Bradyrhizobium japonicum

“…P450s then catalyze subsequent oxidation of ent-kaurene, first to ent-kaurenoic acid in both types of organisms, then in plants to GA 12 and subsequently GA 53 [21], while G. fujikuroi produces GA 14 [22]. To form bioactive nor-diterpenoid (C 19 ) gibberellins, plants then utilize 2ODDs [23], while fungi rely on additional P450s [2,24,25], although formation of the 1,2-double bond of GA 3 in G. fujikuroi is mediated by a desaturase (des) [26]. These clear differences between plant and fungal GA biosynthesis, particularly in enzymatic type (e.g., Fig.…”

Characterization of the Fungal Gibberellin Desaturase as a 2-Oxoglutarate-Dependent Dioxygenase and Its Utilization for Enhancing Plant Growth

“…The formation of GA 3 from ent-kaurene requires the activity of five oxidases (Fig. 1), four of which are cytochrome P450 monooxygenases: P450-4 (ent-kaurene oxidase) oxidizes ent-kaurene to ent-kaurenoic acid , which is converted to GA 14 by P450-1 (GA 14 synthase; Rojas et al, 2001); P450-2 functions as a GA 20-oxidase, converting GA 14 to GA 4 (Tudzynski et al, 2002), while, in the final step of the pathway, P450-3 13-hydroxylates GA 7 to form GA 13 (Tudzynski et al, 2003). However, the nature of the desaturase (DES), which converts GA 4 to GA 7 (Fig.…”