TheTri4 gene ofFusarium sporotrichioides encodes a cytochrome P450 monooxygenase involved in trichothecene biosynthesis

Abstract

TheTri4 gene ofFusarium sporotrichioides was isolated from a cloned DNA fragment carrying theTri5 gene by complementation of aTri4 ⁻ mutant. The nucleotide sequence ofTri4 was determined and the locations of three introns were identified. Analysis ofTri4 mRNA levels revealed that transcription reached maximum levels coincidently with the onset of trichothecene biosynthesis, and then declined 20-fold over the next 8 h. Disruption ofTri4 resulted in the loss of production of both trichothecenes and apotrichodiol and the accumulation of the unoxygenated pathway intermediate trichodiene. Transformants lacking a functionalTri4 gene were able to convert isotrichotriol, an early pathway intermediate, to T-2 toxin suggesting that most pathway enzymes are present inTri4 ⁻ mutants. These data suggest that the enzyme encoded byTri4 catalyzes the first oxygenation step in the trichothecene pathway and participates in apotrichodiol biosynthesis.Tri4 encodes a protein of 520 residues (M _r =59 056) that shows significant homology with members of the superfamily of cytochromes P450. It appears most similar to the CYP3A subfamily (24.6% amino acid identity). Because it contains less than 40% positional identity with other cytochromes P450, theTri4 gene has been placed in a new cytochrome P450 gene family designatedCY P58.