Biosynthesis of Sesqui‐ and Diterpenes by the Gibberellin Producer Fusarium fujikuroi

Abstract

The fungus Fusarium fujikuroi IMI58289 emits a complex pattern of volatile terpenoids including two major compounds, the sesquiterpene alcohol α-acorenol and the diterpene ent-kaurene. ent-Kaurene is the precursor for the phytohormone gibberellic acid (GA(3)) and is produced from geranylgeranyl diphosphate (GGPP) via ent-copalyl diphosphate by the bifunctional ent-copalyl diphosphate/ent-kaurene synthase (CPS/KS). Several structurally related diterpenes were identified as side products of the CPS/KS. Deletion of the cps/ks gene or the whole GA(3) biosynthetic gene cluster resulted in completely abolished diterpene production. Mutants with deletions of the cytochrome P450 monooxygenase gene P450-4, which is responsible for the three oxidation steps from ent-kaurene to ent-kaurenoic acid en route to GA(3), accumulate diterpene hydrocarbons. Feeding with [6,6,6-(2) H(3)] mevalonolactone gave insights into the stereochemistry of the GGPP cyclisation, which operates with a chair-chair-"antipodal" fold. A rational biosynthetic scheme for all identified sesquiterpenes demonstrated their formation from farnesyl diphosphate (FPP) via three alternative initial cyclisations. Genome sequencing revealed the presence of five putative sesquiterpene synthase genes in the F. fujikuroi genome. The structures of several trace compounds from other classes have been identified as new natural products; these were delineated from their mass spectra and unambiguously assigned by comparison to synthetic references.

Two Histone Deacetylases, FfHda1 and FfHda2, Are Important for Fusarium fujikuroi Secondary Metabolism and Virulence

“…Noteworthy, in some cases, all cluster genes showed an altered expression pattern, while in others, only a few genes or even only one single gene of a putative cluster was affected by deletion of ffhda1. Altogether, four and six gene clusters showed differential expression under low-and highnitrogen conditions, respectively (see Table S1 in the supplemental material) (85,86). Four genes of the gibberellin gene cluster (FFUJ_14331, FFUJ_14335, FFUJ_14336, and FFUJ_14337) and four genes of the fumonisin gene cluster (FFUJ_09243, FFUJ_09246, FFUJ_09252, and FFUJ_09254) were significantly downregulated, while only one gene of the cryptic PKS13 mini-gene cluster (FFUJ_12020), consisting most likely only of one to two genes (19), was upregulated under these low-nitrogen conditions (see Table S1 in the supplemental material).…”

Genetic Dissection of Sesquiterpene Biosynthesis by Fusarium fujikuroi

“…Gibberellin biogenesis starts with the formation of ent-kaurene (2) by the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (CPS/KS). [10] Further terpenes from F. fujikuroi are the spirocyclic sesquiterpene aacorenol (3) [11] and the carotenoid neurosporaxanthin (4), which arises from a branch of the b-carotene biosynthetic pathway. [12,13] Fungal sesquiterpene biosynthesis starts from the well-established mevalonate pathway, in which three molecules of acetyl-CoA give the activated C 5 -units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).…”

“…These cascades terminate by simple deprotonation to give sesquiterpene hydrocarbons, or by attack of water to give the corresponding sesquiterpenoid alcohols. [14] We have recently investigated the volatiles of F. fujikuroi IMI58289 by use of a closed-loop stripping apparatus (CLSA) in combination with GC-MS. [11] A representative chromatogram of headspace extract is shown in Figure 1 A. Besides 2, several structurally related diterpenes were identified as side-products of the CPS/KS, by knockout experiments.…”

“…[22] Headspace extracts of this strain contain a much less complex pattern of volatiles, and formation of all diterpenes is abolished in this strain. [11] This strain was selected for the overproduction of sesquiterpenes formed by Ffsc6 or Ffsc4, by complementary introduction of the gpd::ffsc6 or the gpd::ffsc4 overexpression vectors with concomitant deletion of the other gene. The gpd promoter is derived from the glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase and was induced under the growth conditions in glucose-containing medium for terpene production.…”

“…The volatiles emitted by fungal agar plate cultures were collected by use of a CLSA as described previously. [11] Headspace extracts were analyzed by GC-MS on an HP 7890A GC system connected to an HP5975C Mass Selective Detector fitted with a HP-5 MS fused silica capillary column (30 m 0.22 mm i.d., 0.25 mm film; Agilent). Conditions were : inlet pressure, 67 kPa He (23.3 mL min À1 ); injection volume, 1 mL; injector, 250 8C; transfer line, 300 8C; electron energy, 70 eV.…”