Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum

Abstract

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

Functional Analysis of the Fusarielin Biosynthetic Gene Cluster

“…The gene cluster is also preserved in species of Metarhizium [12], Penicillium, and Colletotrichum (Supplementary Table S1). Besides the PKS (FSL1), the remaining genes are predicted to encode a thioesterase (FSL2), an epimerase (FSL3), a cytochrome P450 oxygenase (FSL4), an enoyl reductase (FSL5), and a transcription factor (FSL7).…”

“…FSL6 was excluded from the study as it is unlikely to be part of the conserved fusarielin gene cluster [12]. The generated mutants …”

“…The putative fusarielin gene cluster consists of seven genes (FSL1-7, Table 1) in F. graminearum of which FSL1-5 and FSL7 are predicted to be involved in fusarielin biosynthesis as they are conserved in Aspergillus, where several strain can produce fusarielins A and B [12,13]. The gene cluster is also preserved in species of Metarhizium [12], Penicillium, and Colletotrichum (Supplementary Table S1).…”

“…Fusarielins A-D were first isolated from an unidentified Fusarium strain in a selection procedure for antifungal compounds [11]. Later, fusarielins F, G, and H were isolated from F. graminearum through constitutive expression of the local transcription factor from the responsible gene cluster [12]. …”

Identification and Regulation offusA, the Polyketide Synthase Gene Responsible for Fusarin Production in Fusarium fujikuroi

“…4). The highest similarity to PKS10 is exhibited by PKS9, which is involved in the synthesis of fusarielins and is not related to fusarins (33). The analysis of the predicted GzFUS1 (PKS10) polypeptide with the Simple Modular Architecture Research Tool (SMART [see Materials and Methods]) revealed 10 protein domains: KS, AT, DH, MeT, KR, PP, C, AMP, PP, and NAD (see the legend to Fig.…”