The TRI11 gene of Fusarium sporotrichioides encodes a cytochrome P-450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis

Abstract

Several genes in the trichothecene biosynthetic pathway of Fusarium sporotrichioides have been shown to reside in a gene cluster. Sequence analysis of a cloned DNA fragment located 3.8 kb downstream from TRI5 has led to the identification of the TRI11 gene. The nucleotide sequence of TRI11 predicts a polypeptide of 492 residues (Mr = 55,579) with significant similarity to members of the cytochrome P-450 superfamily. TRI11 is most similar to several fungal cytochromes P-450 (23 to 27% identity) but is sufficiently distinct to define a new cytochrome P-450 gene family, designated CYP65A1. Disruption of TRI11 results in an altered trichothecene production phenotype characterized by the accumulation of isotrichodermin, a trichothecene pathway intermediate. The evidence suggests that TRI11 encodes a C-15 hydroxylase involved in trichothecene biosynthesis.

FIG. 1

Diagram of the trichothecene pathway in F. sporotrichioides. OAc, acetate.

FIG. 2

(A) Restriction map of cosmid 9-1. All of the SacI and SfiI sites in cosmid 9-1 are shown; the only HindIII site shown is the site used to define the fragment cloned into pFSC3-6. SacI fragment 3-3 is also indicated. (B) Map of the trichothecene pathway gene cluster showing the location of pathway gene coding regions. The SacI and HindIII sites of the fragment in pFSC3-6 are shown in addition to the SfiI site. Sa, SacI; Sf, SfiI; H, HindIII.

FIG. 3

Analysis of TRI11 gene disruptants in F. sporotrichioides by PCR. Transformant and NRRL 3299 DNA preparations were used as templates for PCRs primed with oligonucleotides 677 and 678. PCR products were separated on a 1% agarose gel (40 mM Tris-acetate, 1 mM EDTA) and stained with ethidium bromide. Lanes 1 to 5, PCR products from five different transformant DNAs; lane 6, PCR product from F. sporotrichioides NRRL 3299 DNA.

FIG. 4

Chromatogram of F. sporotrichioides culture extracts from a TRI11⁻ transformant (A) and parent strain NRRL 3299 (B). Cultures were grown in GYEP medium for 3 days and analyzed under identical conditions by gas-liquid chromatography as described previously (11). The locations of isotrichodermin and T-2 toxin are indicated.

FIG. 5

Alignments between the conserved heme-binding motifs of TRI11 and other fungal cytochromes P-450. The conserved cysteinyl residues are indicated (boldface). The fungal cytochromes P-450, species, and gene names (and GenBank accession numbers) are as follows: CYP65A1 from F. sporotrichioides, TRI11 (AF011355); CYP60B from Aspergillus nidulans, stcL (U34740); ord1 from A. parasiticus (L40839); CYP57 from N. haematococca, pdm2 (P38364); CYP58 from F. sporotrichioides, TRI4 (U22462); CYP55 from Fusarium oxysporum (M63340); and CYP53 from A. niger, bphA (A22974).