The P450-1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

Recent studies have shown that the genes of the gibberellin (CA) biosynthesis pathway in the fungus Gibberella fujikuroi are organized in a cluster of at least seven genes. P450-1 is one of four cytochrome P450 monooxygenase genes in this cluster. Disruption of the P450-1 gene in the GA-producing wild-type strain IMI 58289 led to total loss of GA production. Analysis of the P450-1-disrupted mutants indicated that CA biosynthesis was blocked immediately after ent-kaurenoic acid. The function of the P450-1 gene product was investigated further by inserting the gene into mutants of G. fujikuroi that lack the entire CA gene cluster; the gene was highly expressed under GA production conditions in the absence of the other GA-biosynthesis genes. Cultures of transformants containing P450-1 converted ent-[C-14]kaurenoic acid efficiently into [C-14]GA(14), indicating that P450-1 catalyzes four sequential steps in the GA-biosynthetic pathway: 7 beta -hydroxylation, contraction of ring B by oxidation at C-6, 3 beta -hydroxylation, and oxidation at C-7. The GA precursors ent-7 alpha -hydroxy[C-14]kaurenoic acid, [C-14]GA(12)- aldehyde, and [C-14]GA(12) were also converted to [C-14]GA(14). In addition, there is an indication that P450-1 may also he involved in the formation of the kaurenolides and fujenoic acids, which are by-products of CA biosynthesis in G. fujikuroi. Thus, P450-1 displays remarkable multifunctionality and may be responsible for the formation of 12 products.