Fusarium Tri4 encodes a key multifunctional cytochrome P450 monooxygenase for four consecutive oxygenation steps in trichothecene biosynthesis

Fusarium Tri4 encodes a cytochrome P450 monooxygenase (CYP) for hydroxylation at C-2 of the first committed intermediate trichodiene (TDN) in the biosynthesis of trichothecenes. To examine whether this CYP further participates in subsequent oxygenation steps leading to isotrichotriol (4), we engineered Saccharomyces cerevisiae for de novo production of the early intermediates by introducing cDNAs of Fusarium graminearum Tri5 (FgTri5 encoding TDN synthase) and Tri4 (FgTri4). From a culture of the engineered yeast grown on induction medium (final pH 2.7), we identified two intermediates, 2alpha-hydroxytrichodiene (1) and 12,13-epoxy-9,10-trichoene-2alpha-ol (2), and a small amount of non-Fusarium trichothecene 12,13-epoxytrichothec-9-ene (EPT). Other intermediates isotrichodiol (3) and 4 were identified in the transgenic yeasts grown on phosphate-buffered induction medium (final pH 5.5-6.0). When Trichothecium roseum Tri4 (TrTri4) was used in place of FgTri4, 4 was not detected in the culture. The three intermediates, 1, 2, and 3, were converted to 4,15-diacetylnivalenol (4,15-diANIV) when fed to a toxin-deficient mutant of F. graminearum with the FgTri4+ genetic background (viz., by introducing a FgTri5- mutation), but were not metabolized by an FgTri4- mutant. These results provide unambiguous evidence that FgTri4 encodes a multifunctional CYP for epoxidation at C-12,13, hydroxylation at C-11, and hydroxylation at C-3 in addition to hydroxylation at C-2.