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

Abstract

The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN , and NADPH binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA 3 , GA 4 , and GA 7 production, but low levels of non-hydroxylated C 20 -GAs (GA 15 and GA 24 ) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs. Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.

Functional Characterization of Two Cytochrome P450 Monooxygenase Genes, P450 - 1 and P450 - 4 , of the Gibberellic Acid Gene Cluster in Fusarium proliferatum ( Gibberella fujikuroi MP-D)

“…One reason for the loss of GA production could also be a mutated gene copy of cpr-Gf, encoding the cytochrome P450 oxidoreductase (23). Deletion of cpr-Gf in the GA-producing strain IMI58289 resulted in a loss of the ability to produce GA 4 , GA 7 , and GA 3 .…”

“…After restriction with NcoI, the promoter was cloned into vector pAS1/NcoI containing the hygromycin resistance marker (25). In cotransformation experiments, either pAN7-1 (hygromycin resistance) (30) or pNR1 (nourseothricin resistance) (18,23) was used for selection of transformants.…”

Cross-Species Hybridization with Fusarium verticillioides Microarrays Reveals New Insights into Fusarium fujikuroi Nitrogen Regulation and the Role of AreA and NMR

“…In addition, nine stress-responsive genes were identified, such as heat shock protein-encoding genes (e.g., the hsp70 gene) and the gene encoding the superoxide dismutase, sod. Finally, the following two glutamine-repressed genes are involved in secondary metabolism: cpr, encoding the NADPHcytochrome P450 oxidoreductase donating electrons to the P450 enzymes from the GA biosynthetic pathway (37), and bik3, encoding the bikaverin-specific O-methyltransferase (Wiemann and Tudzynski, unpublished). A total of 33% share homology to genes with no known function.…”

Cassava Superelongation Disease in the Caribbean

“…Although plants and fungi produce structurally identical gibberellins, the biosynthetic steps in the pathway for the formation of gibberellins differs significantly [36]. A major contrast in the biosynthetic pathway is the stage at which the hydroxyl groups are introduced.…”