Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum

Abstract

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

Keywords: FAD-oxidase; acetyltransferase; cytochrome P450 oxidoreductase; emerging mycotoxins; fusaproliferin; knock-out; preterpestacin; terpenoid synthase; terpestacin.

Figure 1

PCR confirmation of the knock-out of FUP1 and FUP3 to FUP5. (A) screening scheme with primer locations indicated; (B) screening of FUP1 knock out using multiplex PCR (Primer A + C + E or B + D + F). The red arrows indicate the expected length of the knock-out band; (C) length of the expected fragments is shown in Table 2; (D–F) screening with two primers. For the samples on the top panels the wild-type primer pair (A + C or B + D) was used, on the bottom the pair specific for the knock-out (A + E or B + F) was used; (D) screening for FUP3 knock-out (E) screening for FUP4 knock out; (F) screening for FUP5 knock out; (D–F) the red arrows beside the respective gel indicate the expected lengths. Size standard: 1 kb ladder (Thermo Fisher Scientific, Vienna, Austria).

Figure 2

Extracted ion chromatograms of putative FUP biosynthetic pathway compounds from a sample mixture which consisted of equal volume aliquots of each sample type. [M+H]+ adducts were considered for compounds 1, 2, 3, 8, 9 and [M-H₂O+H]+ for 4, 5, 6, 7, 10, 11. For visualization of all compounds in a single overlaid chromatogram, the peak abundances of 8, 4 and 11 are divided by 10.

Figure 3

Scheme which shows the intermediates of the proposed FUP biosynthetic pathway with side reactions of pathway intermediates, as well as their abundance distribution in samples of Fusarium proliferatum wild-type (WT) and knock-out strains. Thick arrows correspond to reactions of the proposed pathway, thin arrows to side reactions of the pathway intermediates. Dashed arrows indicate reactions that may take place but were not proved with the present experiment.

Figure 3

Scheme which shows the intermediates of the proposed FUP biosynthetic pathway with side reactions of pathway intermediates, as well as their abundance distribution in samples of Fusarium proliferatum wild-type (WT) and knock-out strains. Thick arrows correspond to reactions of the proposed pathway, thin arrows to side reactions of the pathway intermediates. Dashed arrows indicate reactions that may take place but were not proved with the present experiment.

Figure 4

Molecular network of FUP, TPC and suspect compounds. Compound numbers in nodes correspond to those from Table 4. Two nodes are connected over the edge if their cosine score is higher than 0.5. Edges that have a cosine score higher than 0.6 have a weighted thickness that increases with the score value.