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

• Published in: Biochemical and biophysical research communications 2007-02, Vol.353 (2), p.412-417
• Main Authors: Tokai, Takeshi, Koshino, Hiroyuki, Takahashi-Ando, Naoko, Sato, Masayuki, Fujimura, Makoto, Kimura, Makoto
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; BIOSYNTHESIS; Biosynthesis gene cluster; CARBON 11; CARBON 12; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; EPOXIDES; Fungal secondary metabolites; FUSARIUM; Fusarium - genetics; Fusarium - metabolism; Fusarium graminearum; GENES; HYDROXYLATION; METABOLITES; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Multienzyme Complexes - metabolism; Multifunctional cytochrome P450 monooxygenase; MUTANTS; MUTATIONS; Oxidation-Reduction; Oxygen - metabolism; PATHOGENS; PH VALUE; PHOSPHATES; Plant pathogen; Promoter Regions, Genetic - genetics; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Signal Transduction - physiology; TOXINS; Trichothecene mycotoxin; Trichothecenes - biosynthesis; VIRULENCE; Virulence factor
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2006.12.033

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, 2α-hydroxytrichodiene ( 1) and 12,13-epoxy-9,10-trichoene-2α-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.