Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi

Abstract

Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.

Mechanistic investigations on six bacterial terpene cyclases

“…These experiments demonstrate that in case of all six sesquiterpene cyclases the substrate FPP is converted with a strict stereochemical course with respect to the fate of the diastereotopic methyl groups in FPP. These findings are in agreement with those reported for several other terpene cyclases [58–60], while the (1 R ,4 R ,5 S )-guaia-6,10(14)-diene synthase from Fusarium fujikuroi shows a relaxed stereochemical course in this aspect [61]. …”

Comparative “Omics” of theFusarium fujikuroiSpecies Complex Highlights Differences in Genetic Potential and Metabolite Synthesis

“…S6, Supplementary Material online). Recently, the products of STC3 and STC5 were identified by heterologous expression of F. fujikuroi STC3 and F. mangiferae STC5 in Escherichia coli as eremophilene and guaia-6,10(14)-diene, respectively (Burkhardt et al 2016). The failure to detect the product of F. fujikuroi STC5 was likely due to a point mutation in the conserved NSE triad motif (supplementary fig.…”

“…S7, Supplementary Material online). Alternation of the F. fujikuroi STC5 NSE triad motif by site directed mutagenesis led to the production of guaia-6,10(14)-diene (Burkhardt et al 2016). Pseudogenization also likely played a role in the failure to detect products of the other F. fujikuroi STCs.…”

“…7; supplementary table S3, Supplementary Material online). STC5 , which is responsible for production of guaia-6,10(14)-diene (Burkhardt et al 2016), was expressed only in F. proliferatum ET1 on CM and at 20 dpi.…”

Volatile communication between fungi and bacteria

“…The initial products can be further converted into highly functionalised and bioactive molecules such as mycotoxins by oxidations, acylations and other modifications. In the past two decades, several fungal terpene cyclases and their related products have been characterised, including the sesquiterpene cyclases for (-)-(1R,4R,5S)-guaia-6,10(14)-diene, (+)-eremophilene, (+)-koraiol and (-)-α-acorenol from Fusarium fujikuroi (Brock et al, 2013, Burkhardt et al, 2016, longiborneol from F. graminearum (McCormick et al, 2010), trichodiene from F.…”