Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs.

Background and aims The extent to which genome size and chromosome numbers evolve in concert is little understood, particularly after polyploidy (whole-genome duplication), when a genome returns to a diploid-like condition (diploidisation). We study this phenomenon in 46 species of allotetraploid Nicotiana section Suaveolentes (Solanaceae), which formed less than six million years ago and radiated in the arid centre of Australia. Methods We analysed newly assessed genome sizes and chromosome numbers within the context of a restriction site-associated nuclear DNA (RADseq) phylogenetic framework. Key results RADseq generated a well-supported phylogenetic tree, in which multiple accessions from each species formed unique genetic clusters. Chromosome numbers and genome sizes vary from n = 2x = 15-24 and 2.7-5.8 pg/1 C nucleus, respectively. Decreases in both genome size and chromosome number occur, although neither consistently nor in parallel. Species with the lowest chromosome numbers (n = 15–18) do not possess the smallest genome sizes, and although N. heterantha has retained the ancestral chromosome complement, n = 2x = 24, it nonetheless has the smallest genome size, even smaller than that of the modern representatives of ancestral diploids. Conclusions The results indicate that decreases in genome size and chromosome number occur in parallel down to a chromosome number threshold, n = 20, below which genome size increases, a phenomenon potentially explained by decreasing rates of recombination over fewer chromosomes. We hypothesize that, more generally in plants, major decreases in genome size post-polyploidization take place while chromosome numbers are still high because in these stages elimination of retrotransposons and other repetitive elements is more efficient. Once such major genome size change has been accomplished, then dysploid chromosome reductions take place to reorganize these smaller genomes, producing species with small genomes and low chromosome numbers such as those observed in many annual angiosperms, including Arabidopsis.

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Background: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a highly diverse group of secondary metabolites (SM) of bacterial and fungal origin. While RiPPs have been intensively studied in bacteria, little is known about fungal RiPPs. In Fungi only six classes of RiPPs are described. Current strategies for genome mining are based on these six known classes. However, the genes involved in the biosynthesis of theses RiPPs are normally organized in biosynthetic gene clusters (BGC) in fungi. Results: Here we describe a comprehensive strategy to mine fungal genomes for RiPPs by combining and adapting existing tools (e.g. antiSMASH and RiPPMiner) followed by extensive manual curation based on conserved domain identification, (comparative) phylogenetic analysis, and RNASeq data. Deploying this strategy, we could successfully rediscover already known fungal RiPPs. Further, we analysed four fungal genomes from the Trichoderma genus. We were able to find novel potential RiPP BGCs in Trichoderma using our unconventional mining approach. Conclusion: We demonstrate that the unusual mining approach using tools developed for bacteria can be used in fungi, when carefully curated. Our study is the first report of the potential of Trichoderma to produce RiPPs, the detected clusters encode novel uncharacterized RiPPs. The method described in our study will lead to further mining efforts in all subdivisions of the fungal kingdom.

Background Aspergillus niger is a ubiquitous filamentous fungus widely employed as a cell factory thanks to its abilities to produce a wide range of organic acids and enzymes. Its genome was one of the first Aspergillus genomes to be sequenced in 2007, due to its economic importance and its role as model organism to study fungal fermentation. Nowadays, the genome sequences of more than 20 A. niger strains are available. These, however, do not include the neotype strain CBS 554.65. Results The genome of CBS 554.65 was sequenced with PacBio. A high-quality nuclear genome sequence consisting of 17 contigs with a N50 value of 4.07 Mbp was obtained. The assembly covered all the 8 centromeric regions of the chromosomes. In addition, a complete circular mitochondrial DNA assembly was obtained. Bioinformatic analyses revealed the presence of a MAT1-2-1 gene in this genome, contrary to the most commonly used A. niger strains, such as ATCC 1015 and CBS 513.88, which contain a MAT1-1-1 gene. A nucleotide alignment showed a different orientation of the MAT1–1 locus of ATCC 1015 compared to the MAT1–2 locus of CBS 554.65, relative to conserved genes flanking the MAT locus. Within 24 newly sequenced isolates of A. niger half of them had a MAT1–1 locus and the other half a MAT1–2 locus. The genomic organization of the MAT1–2 locus in CBS 554.65 is similar to other Aspergillus species. In contrast, the region comprising the MAT1–1 locus is flipped in all sequenced strains of A. niger. Conclusions This study, besides providing a high-quality genome sequence of an important A. niger strain, suggests the occurrence of genetic flipping or switching events at the MAT1–1 locus of A. niger. These results provide new insights in the mating system of A. niger and could contribute to the investigation and potential discovery of sexuality in this species long thought to be asexual.

Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis . Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approx. 59 Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, several of which could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon-optimized and expressed, together with farnesyl-, geranyl- and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. Analysis of recombinant strains revealed production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. I mportance Endophytic fungi represent as yet underexplored source of secondary metabolites, some of which may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used both cultivation of the isolate with subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed enormous potential of this Helotiales isolate to produce structurally diverse natural products, including polyketides, non-ribosomally synthesized peptides, terpenoids and RiPPs. Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes sets a stage for investigation of the respective biosynthetic pathways. Heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.

In this work, we present the whole-genome sequence and the complete mitochondrial sequence of the black yeast-like strain Aureobasidium pullulans var. aubasidani CBS 100524, which produces the exopolysaccharide aubasidan and was previously isolated from Betula sp. slime flux from the Leningrad Region of Russia.

Background The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. Results We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. Conclusion The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.