Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Abstract

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated ␥-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to-aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.The plant pathogenic fungus Fusarium graminearum (teleomorph Gibberella zeae) is capable of producing a plethora of secondary metabolites, many of which belong to the polyketide class of compounds, such as the mycotoxins zearalenone, fusarin C, and aurofusarin (1). The F. graminearum genome encodes 15 polyketide synthases (PKS) 2 (2, 3), of which FgPKS12 is the progenitor of nor-rubrofusarin/rubrofusarin/aurofusarin (4, 5), FgPKS4 and FgPKS13 are the progenitors of zearalenone (1, 6), FgPKS10 is the progenitor of fusarin C (7, 8), and FgPKS3 is the progenitor of an uncharacterized black/purple perithecial pigment (8). PKS genes are typically found in gene clusters that comprise genes encoding transcription factor, transporters, and tailoring enzymes required for biosynthesis of the final product (1, 4 -6). In addition to genes encoding well characterized classes of enzymes, the clusters typically also include orphan genes that encode proteins annotated as "hypothetical" or "conserved hypothetical proteins," signifying that no similarity to any previously characterized protein has been found. In the case of F. graminearum, more than half of all gene models found in the Fusarium graminearum Genome Database from the Munich Information Center for Protein Sequences (MIPS) fall into either of these two categories (9).Functional characterization of hypothetical proteins is challenging, but those belonging to secondary metabolite gene clusters provide a unique opportunity because their location suggests a function in the respective biosynthetic pathways. The PKS12 gene cluster in F. graminearum, which is responsible for biosyn...

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

“…pks4 belongs to the nonreducing clade I of fungal pks-encoding genes, which includes genes associated with pigment production, such as aurofusarin (7)(8)(9) and bikaverin (10,11), but also DHN melanin (12)(13)(14)(15)(16)(17)(18)(19). While the former comprise substances of relatively low molecular weights, melanins-the dark to black pigments-are of high molecular mass that derive from oxidative polymerization of phenolic compounds (20,37).…”

“…A total of 11 PKS-encoding genes were found in the T. reesei genome, among which 2 occur only in T. reesei and 9 have orthologues in T. virens or/and T. atroviride PKSs (6). pks4 (Trire2: 82208, Triat2:79, and Trive2:77826 in T. reesei, T. atroviride, and T. virens, respectively), which encodes an enzyme of the nonreducing type (clade I), has been shown to have orthologues in other fungi, i.e., PKSs associated with synthesis of aurofusarin in Fusarium graminearum (7)(8)(9), bikaverin in F. fujikuroi (10,11), and DHN melanin in Aspergillus spp. (12)(13)(14)(15)(16)(17)(18)(19).…”

Characterization of a fungal thioesterase having claisen cyclase and deacetylase activities in melanin biosynthesis

“…The protein sequence of Pks1 was compared to other NR-PKSs involved in melanin and conidial pigment biosynthesis. Several YWA1-producing PKSs have been functionally characterized including A. fumigatus Alb1 , A. nidulans wA , A. niger FwnA (Chiang, et al, 2011;Jorgensen, et al, 2011), and Gibberella zeae Pks12 (Frandsen, et al, 2011). WdPks1 from W. dermatitidis is the sole validated example of an ATHN synthase (Wheeler, et al, 2008).…”

Phylogenomic analysis of polyketide synthase-encoding genes in Trichoderma

“…The genomes of all three Trichoderma genomes encode PKS genes that group in the non-reducing fungal PKS clade I, which includes the genes associated with Fusarium graminearum aurofusarin (Frandsen et al, 2011;Kim et al, 2005;Malz et al, 2005), Fusarium fujikuroi bikaverin (Linnemannstöns et al, 2002;Wiemann et al, 2009) and Aspergillus spp. DHN melanin (Baker, 2008; Chiang et al, 2011;Jørgensen et al, 2011; Langfelder et al, 1998; Tsai et al, 1998Tsai et al, , 2001Watanabe et al, 1999Watanabe et al, , 2000.…”

“…3). The third gene in the putative pigment clusters shows amino acid sequence similarity to the recently characterized protein product of F. graminearum aurZ, which has been shown to convert the compound YWA1 to nor-rubofusarin (Frandsen et al, 2011). None of the Trichoderma genomes has genes that are related to known hydroxynaphthalone (HN) reductases, which catalyse the conversion of 1,3,6,8-tetrahydroxynaphthalene to scytalone and the reduction of 1,3,8-trihydroxynaphthalene to vermelone.…”