Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style.

Summary Filamentous fungi produce a vast array of secondary metabolites (SMs) and some play a role in agriculture or pharmacology. Sequencing of the rice pathogen Fusarium fujikuroi revealed the presence of far more SM-encoding genes than known products. SM production is energy-consuming and thus tightly regulated, leaving the majority of SM gene clusters silent under laboratory conditions. One important regulatory layer in SM biosynthesis involves histone modifications that render the underlying genes either silent or poised for transcription. Here, we show that the majority of the putative SM gene clusters in F. fujikuroi are located within facultative heterochromatin marked by trimethylated lysine 27 on histone 3 (H3K27me3). Kmt6, the methyltransferase responsible for establishing this histone mark, appears to be essential in this fungus, and knock-down of Kmt6 in the KMT6kd strain shows a drastic phenotype affecting fungal growth and development. Transcription of four so far cryptic and otherwise silent putative SM gene clusters was induced in the KMT6kd strain, in which decreased expression of KMT6 is accompanied by reduced H3K27me3 levels at the respective gene loci and accumulation of novel metabolites. One of the four putative SM gene clusters, named STC5, was analysed in more detail thereby revealing a novel sesquiterpene.

Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety.

Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.

Octocorals are major contributors of terpenoid chemical diversity in the ocean. Natural products from other sessile marine animals are primarily biosynthesized by symbiotic microbes rather than by the host. Here we challenge this long-standing paradigm by describing a monophyletic lineage of animal-encoded terpene cyclases (TCs), ubiquitous in octocorals. We characterized 15 TC enzymes from nine genera, several of which produce precursors of iconic, coral-specific terpenoids such as pseudopterosin, lophotoxin and eleutherobin. X-ray crystallography reveals that coral TCs share conserved active site residues and structural features with bacterial TCs. The identification of coral TCs enabled the targeted identification of the enzyme that constructs the coral-exclusive capnellane scaffold. Several TC genes are co-localized with genes that encode enzymes known to modify terpenes. This work presents the first example of biosynthetic capacity in the kingdom Animalia that rivals the chemical complexity generated by plants, unlocking the biotechnological potential of octocorals for biomedical applications.