Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 May 8;98(10):5838-43.
doi: 10.1073/pnas.091096298. Epub 2001 Apr 24.

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

M C Rojas  1 , P Hedden, P Gaskin, B Tudzynski
Affiliations

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

M C Rojas et al. Proc Natl Acad Sci U S A. .

Abstract

Recent studies have shown that the genes of the gibberellin (GA) 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 GA 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 GA 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-[(14)C]kaurenoic acid efficiently into [(14)C]GA(14), indicating that P450-1 catalyzes four sequential steps in the GA-biosynthetic pathway: 7beta-hydroxylation, contraction of ring B by oxidation at C-6, 3beta-hydroxylation, and oxidation at C-7. The GA precursors ent-7alpha-hydroxy[(14)C]kaurenoic acid, [(14)C]GA(12)-aldehyde, and [(14)C]GA(12) were also converted to [(14)C]GA(14). In addition, there is an indication that P450-1 may also be involved in the formation of the kaurenolides and fujenoic acids, which are by-products of GA biosynthesis in G. fujikuroi. Thus, P450-1 displays remarkable multifunctionality and may be responsible for the formation of 12 products.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Biosynthetic pathways to GAs and ent-kaurenoids in G. fujikuroi, indicating the reactions we propose to be catalyzed by P450–1 (between dotted lines). Thick arrows depict proposed major pathway from ent-kaurenoic acid to GA14.
Figure 2
Figure 2
The P450–1 locus in the GA gene cluster in G. fujikuroi. (A) Physical map of the P450–1 ORF and surrounding genomic region. (B) Construction of the gene replacement vector pP450–1-GR.
Figure 3
Figure 3
Molecular analysis of P450–1 replacement mutants (A) Southern blot analysis of G. fujikuroi strains IMI 58289 (wild type), SG139 (mutant lacking GA-biosynthesis gene cluster), and gene-disruption mutants ΔP450–1-T9 and ΔP450–1-T35. Genomic DNA from each strain was digested with either HindIII (lanes 1–3) or PstI (lanes 4–6), and the blots were probed with P450–1 cDNA. The size standards are indicated in kilobases. (B) Northern blot analysis of the wild-type strain IMI 58289 and the replacement mutants T9 and T35. The blot was probed with the P450–1 cDNA and the Botrytis cinerea actin gene (loading control)
Figure 4
Figure 4
Characterization of gene disruption mutant ΔP450–1-T35. (A) Total ion currents from GC/MS analyses of extracts of culture filtrates and mycelia from G. fujikuroi strains IMI 58289 (wild type) and ΔP450–1-T35. (B and C) HPLC radiochromatograms of extracts from the culture filtrates and mycelia after incubation of ent-[14C4]kaurenoic acid (B) and [14C4]GA12-aldehyde (culture filtrate only) (C) with IMI58289 and ΔP450–1-T35.
Figure 5
Figure 5
Northern blot analysis of the G. fujikuroi deletion mutants SG139 and Gib1, and strains transformed with the vector P450–1-GC carrying the complete P450–1 gene copy. The blot was probed with the P450–1 cDNA and the Botrytis cinerea actin gene (loading control)
Figure 6
Figure 6
HPLC-radioactivity profiles of total extracts from incubations of ent-[14C4]kaurenoic acid, ent-7α-hydroxy[14C4]kaurenoic acid, [14C4]GA12-aldehyde, or [14C4]GA12 with the G. fujikuroi wild-type strain IMI 58289, deletion mutant SG139, and the P450–1 transformant SG139-T32. Retention times for ent-kaurenoic acid, ent-7α-hydroxykaurenoic acid, GA12-aldehyde, and GA12 are 22, 16, 18, and 17 min, respectively. HPLC and incubation conditions were as described in Materials and Methods, except for ent-kaurenoic acid with SG139-T32, for which the incubation time was 4 h.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hooley R. Plant Mol Biol. 1994;26:1529–1555. - PubMed
    1. Rademacher W. Plant Growth Regul. 1994;15:303–314.
    1. MacMillan J. Nat Prod Res. 1997;14:221–243.
    1. Tudzynski B. In: The Mycota V, Plant Relationship, Part A. Esser K, Lemke P A, editors. Berlin: Springer; 1997. pp. 167–184.
    1. Hedden P, Phillips A L. Trends Plant Sci. 2000;5:523–530. - PubMed

Publication types

LinkOut - more resources