Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

Marine Fungi are potent secondary metabolite producers. However, limited genetic information are available their biosynthetic gene clusters (BGCs) and their biotechnological applications. To overcome this lack of information, herein, we used next-generation sequencing methods for genome sequencing of two marine fungi, isolated from the German Wadden Sea, namely Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079. The assembled genome size of the marine isolate Calcarisporium sp. KF525 is about 36.8 Mb with 60 BGCs, while Pestalotiopsis sp. KF079 has a genome size of 47.5 Mb harboring 67 BGCs. Of all BGCs, 98% and 97% are novel clusters of Calcarisporium sp. and Pestalotiopsis sp., respectively. Only few of the BGCs were found to be expressed under laboratory conditions by RNA-seq analysis. The vast majority of all BGCs were found to be novel and unique for these two marine fungi. Along with a description of the identified gene clusters, we furthermore present important genomic features and life-style properties of these two fungi. The two novel fungal genomes provide a plethora of new BGCs, which may have biotechnological applications in the future, for example as novel drugs. The genomic characterizations will provide assistance in future genetics and genomic analyses of marine fungi.

Flowering is a crucial stage during plant development. Plants may respond to unfavorable conditions by accelerating reproductive processes like flowering. In a recent study, we showed that PRECOCIOUS1 (POCO1) is a mitochondrial pentatricopeptide repeat (PPR) protein involved in flowering time and abscisic acid (ABA) signaling in Arabidopsis thaliana. Here, we use RNA-seq data to investigate global gene expression alteration in the poco1 mutant.

The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Red yeasts of the genus Rhodotorula are of great interest to the biotechnological industry due to their ability to produce valuable natural products, such as lipids and carotenoids with potential applications as surfactants, food additives, and pharmaceuticals. Herein, we explored the biosynthetic potential of R. mucilaginosa 50-3-19/20B collected from the Mid-Atlantic Ridge using modern genomics and untargeted metabolomics tools. R. mucilaginosa 50-3-19/20B exhibited anticancer activity when grown on PDA medium, while antimicrobial activity was observed when cultured on WSP-30 medium. Applying the bioactive molecular networking approach, the anticancer activity was linked to glycolipids, namely polyol esters of fatty acid (PEFA) derivatives. We purified four PEFAs (1-4) and the known methyl-2-hydroxy-3-(1H-indol-2-yl)propanoate (5). Their structures were deduced from NMR and HR-MS/MS spectra, but 1-5 showed no anticancer activity in their pure form. Illumina-based genome sequencing, de novo assembly and standard biosynthetic gene cluster (BGC) analyses were used to illustrate key components of the PEFA biosynthetic pathway. The fatty acid producing BGC3 was identified to be capable of producing precursors of PEFAs. Some Rhodotorula strains are able to convert inulin into high-yielding PEFA and cell lipid using a native exo-inulinase enzyme. The genomic locus for an exo-inulinase enzyme (g1629.t1), which plays an instrumental role in the PEFA production via the mannitol biosynthesis pathway was identified. This is the first untargeted metabolomics study on R. mucilaginosa providing new genomic insights into PEFA biosynthesis.

Plants, bacteria and some fungi are known to produce indole-3-acetic acid (IAA) by employing various pathways. Among these pathways, the indole-3-pyruvic acid (IPA) pathway is the best studied in green plants and plant-associated beneficial microbes. While IAA production circuitry in plants has been studied for decades, little is known regarding the IAA biosynthesis pathway in fungal species. Here, we present the first data for IAA-producing genes and the associated biosynthesis pathway in a non-pathogenic fungus, Neurospora crassa. For this purpose, we used a computational approach to determine the genes and outlined the IAA production circuitry in N. crassa. We then validated these data with experimental evidence. Here, we describe the homologous genes that are present in the IPA pathway of IAA production in N. crassa. High-performance liquid chromatography and thin-layer chromatography unambiguously identified IAA, indole-3-lactic acid (ILA) and tryptophol (TOL) from cultures supplemented with tryptophan. Deletion of the gene (cfp) that encodes the enzyme indole-3-pyruvate decarboxylase, which converts IPA to indole-3-acetaldehyde (IAAld), results in an accumulation of higher levels of ILA in the N. crassa culture medium. A double knock-out strain (Δcbs-3;Δahd-2) for the enzyme IAAld dehydrogenase, which converts IAAld to IAA, shows a many fold decrease in IAA production compared with the wild type strain. The Δcbs-3;Δahd-2 strain also displays slower conidiation and produces many fewer conidiospores than the wild type strain.

As previously demonstrated, the maize cox2 RNA is fully edited in cauliflower mitochondria. Use of constructs with a deleted cox2 intron, however, led to a loss of RNA editing at almost all editing sites, with only a few sites still partially edited. Likewise, one deletion in exon 1 and three in exon 2 abolish RNA editing at all cox2 sites analyzed. Furthermore, intron splicing is abolished using these deletions. Mutation of a cytosine residue, which is normally edited and localized directly adjacent to the intron, to thymidine did not result in restoration of splicing, indicating that the loss of splicing was not due to loss of RNA editing. One deletion in exon 2 did not lead to loss of splicing. Instead, most editing sites were found to be edited, only three were not edited. Unexpectedly, we observed additional RNA editing events at new sites. Thus it appears that deletions in the cox2 RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. As these effects are not limited to the vicinity of the respective deletions, but appear to be widespread or even affect all editing sites, they may not be explained by the loss of PPR binding sites. Instead, it appears that several parts of the cox2 transcript are required for proper RNA processing. This indicates the roles of the RNA sequence and structural elements in the recognition of the editing sites.

MCC/eisosomes are protein-organized domains in the plasma membrane of fungi and algae. However, the composition and function(s) of MCC/eisosomes in the filamentous fungus Neurospora crassa were previously unknown. To identify proteins that localize to MCC/eisosomes in N. crassa, we isolated proteins that co-purified with the core MCC/eisosome protein LSP-1, which was tagged with GFP. Proteins that co-fractionated with LSP-1:GFP were then identified by mass spectrometry. Eighteen proteins were GFP-tagged and used to identify six proteins that highly colocalized with the MCC/eisosome marker LSP-1:RFP, while five other proteins showed partial overlap with MCC/eisosomes. Seven of these proteins showed amino acid sequence homology with proteins known to localize to MCC/eisosomes in the yeast Saccharomyces cerevisiae. However, homologs of three proteins known to localize to MCC/eisosomes in S. cerevisiae (Can1, Pkh1/2, and Fhn1) were not found to colocalize with MCC/eisosome proteins in N. crassa by fluorescence microscopy. Interestingly, one new eisosome protein (glutamine-fructose-6-phosphate aminotransferase, gene ID: NCU07366) was detected in our studies. These findings demonstrate that there are interspecies differences of the protein composition of MCC/eisosomes. To gain further insight, molecular modeling and bioinformatics analysis of the identified proteins were used to propose the organization of MCC/eisosomes in N. crassa. A model will be discussed for how the broad range of functions predicted for the proteins localized to MCC/eisosomes, including cell wall synthesis, response and signaling, transmembrane transport, and actin organization, suggests that MCC/eisosomes act as organizing centers in the plasma membrane.

Eisosomes are plasma-membrane-associated protein complexes of fungi and algae involved in various cellular processes. The eisosome composition of the budding yeast is well described, but there is a limited number of studies only about eisosomes in filamentous fungi. In our study, we examined the Neurospora crassa LSP-1 protein (NcLSP1). By complementing a Saccharomyces cerevisiae Δpil1 mutant strain with nclsp1, we show the functional homology of the NcLSP1 to yeast PIL1 rather than to yeast LSP1 and hereby confirm that the NcLSP1 is an eisosomal core protein and suitable eisosomal marker. The subsequent cloning and expression of the nclsp1::trfp reporter gene construct in N. crassa allowed for a systematical investigation of the characteristics of eisosome formation and distribution in different developmental stages. In N. crassa, the hyphae germinating from sexual and asexual spores are morphologically identical and have been historically recognized as the same type of cells. Here, we demonstrate the structural differences on the cellular level between the hyphae germinating from sexual and asexual spores.

Fungi are a large group of eukaryotes found in nearly all ecosystems. More than 250 fungal genomes have already been sequenced, greatly improving our understanding of fungal evolution, physiology, and development. However, for the Pezizomycetes, an early-diverging lineage of filamentous ascomycetes, there is so far only one genome available, namely that of the black truffle, Tuber melanosporum, a mycorrhizal species with unusual subterranean fruiting bodies. To help close the sequence gap among basal filamentous ascomycetes, and to allow conclusions about the evolution of fungal development, we sequenced the genome and assayed transcriptomes during development of Pyronema confluens, a saprobic Pezizomycete with a typical apothecium as fruiting body. With a size of 50 Mb and ~13,400 protein-coding genes, the genome is more characteristic of higher filamentous ascomycetes than the large, repeat-rich truffle genome; however, some typical features are different in the P. confluens lineage, e.g. the genomic environment of the mating type genes that is conserved in higher filamentous ascomycetes, but only partly conserved in P. confluens. On the other hand, P. confluens has a full complement of fungal photoreceptors, and expression studies indicate that light perception might be similar to distantly related ascomycetes and, thus, represent a basic feature of filamentous ascomycetes. Analysis of spliced RNA-seq sequence reads allowed the detection of natural antisense transcripts for 281 genes. The P. confluens genome contains an unusually high number of predicted orphan genes, many of which are upregulated during sexual development, consistent with the idea of rapid evolution of sex-associated genes. Comparative transcriptomics identified the transcription factor gene pro44 that is upregulated during development in P. confluens and the Sordariomycete Sordaria macrospora. The P. confluens pro44 gene (PCON_06721) was used to complement the S. macrospora pro44 deletion mutant, showing functional conservation of this developmental regulator.