Genetic Manipulation of the Fusarium fujikuroi Fusarin Gene Cluster Yields Insight into the Complex Regulation and Fusarin Biosynthetic Pathway

Abstract

In this work, the biosynthesis and regulation of the polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS)-derived mutagenic mycotoxin fusarin C was studied in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes (fus1-fus9) that are coexpressed under high-nitrogen and acidic pH conditions. Chromatin immunoprecipitation revealed a correlation between high expression and enrichment of activating H3K9-acetylation marks under inducing conditions. We provide evidence that only four genes are sufficient for the biosynthesis. The combination of genetic engineering with nuclear magnetic resonance and mass-spectrometry-based structure elucidation allowed the discovery of the putative fusarin biosynthetic pathway. Surprisingly, we indicate that PKS/NRPS releases its product with an open ring structure, probably as an alcohol. Our data indicate that 2-pyrrolidone ring closure, oxidation at C-20, and, finally, methylation at C-20 are catalyzed by Fus2, Fus8, and Fus9, respectively.

Expression Analysis of PKS13, FG08079.1 and PKS10 Genes in Fusarium graminearum and Fusarium culmorum

“…It was reported that FUS C was actively synthesized during growing mycelia, under high nitrogen concentrations or acidic conditions (8,16). Therefore, the provided condition most likely was not suitable to induce the expression of PKS10.…”

Fusarins and Fusaric Acid in Fusaria

“…By combining single and multiple gene deletions, overexpression of cluster genes and co-cultivation of knockout mutants with different genetic blocks, Niehaus et al recently provided evidence that only four of the nine co-regulated cluster genes ( FUS1 , FUS2 , FUS8 , and FUS9 ) are essential for the production of fusarins [ 73 ]. The PKS part of the hybrid PKS/NRPS enzyme is responsible for the condensation of one acetyl CoA and six malonyl CoA units [ 87 ].…”

“…Only after this oxidation step, Fus2 catalyzes the formation of the 2-pyrrolidone ring what enables the P450 monooxygenase Fus8 to catalyze additional oxidation steps at the C-20 atom. At last, the methyltransferase Fus9 methylates the hydroxy group at C-21 and forms fusarin C [ 73 ] (Fig. 11.3 ).…”

“…The PKS/NRPS forms the amide linkage between the heptaketide part of the PKS and the activated homoserine of the NRPS part [ 114 ]. The PKS/ NRPS product is released as an alcohol, prefusarin, which is then hydroxylated by the P450 monooxygenase Fus8 at carbon C-20 to 20-hydroxy-fusarin ( [ 73 ]). Only after this oxidation step, Fus2 catalyzes the formation of the 2-pyrrolidone ring what enables the P450 monooxygenase Fus8 to catalyze additional oxidation steps at the C-20 atom.…”

“…Recently, the entire fusarin gene cluster consisting of nine co-regulated genes ( FUS1 -FUS9 ) has been characterized in F. fujikuroi . However, only four of them ( FUS1 , FUS2 , FUS8 , and FUS9 ) are involved in the biosynthesis of the fusarins [ 73 ]. FUS1 encodes the key enzyme, the hybrid PKS/NRPS.…”