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 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 2alpha-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.
Highlights
Gibberellins (GAs)[1] are plant hormones that are produced by sis gene cluster in Gibberella fujikuroi were cloned and all higher plants and some fungi
Most of the genes of the early isoprenoid pathway have been cloned from G. fujikuroi, viz 3-hydroxy-3-methylglutaryl-CoA reductase (2), farnesyl diphosphate synthase (3), and two geranylgeranyl diphosphate (GGPP) synthase genes, one of which is involved in general isoprenoid biosynthesis (4) and a second of which is specific for GA biosynthesis (5)
GGPP is converted to ent-kaurene via ent-copalyldiphosphate in a two-step cyclization reaction (6). ent-Kaurene is metabolized to GAs in G. fujikuroi by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenases, whereas in plants, P450 monooxygenases and 2-oxoglutaratedependent dioxygenases are involved (7)
Summary
Gibberellins (GAs)[1] are plant hormones that are produced by sis gene cluster in Gibberella fujikuroi were cloned and all higher plants and some fungi. For expression of orf[3] and P450-3 in the GA-deficient mutant SG139, 107 protoplasts were transformed with 10 g of the complementation vectors porf3-GC, pP450 –3-GC, or the cosmid clone GA-cos[5], carrying the entire P450 –3 gene and an ϳ40-kb genomic region to the right of P450 –3 probably not involved in GA biosynthesis.
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