Higher yields of cyclodepsipetides from Scopulariopsis brevicaulis by random mutagenesis

The ascomycete Scopulariopsis brevicaulis, which was isolated from the marine sponge Tethya aurantium, produces two cyclodepsipeptides, scopularides A and B [1]. Both peptides exhibit activity against several tumor cell lines. Within the EU-project MARINE FUNGI (EU FP7, 265926) one of our aims is to enhance the production of these secondary metabolites. We are in the process to establish two ways of random mutagenesis by both UV radiation and transposon-mediated. To this end we created UV-mutants and a miniaturised screening method was developed. UV-radiation was performed at 312 nm and the survival rate was set to 1 %. With this method a mutant library was established. To screen these mutants for higher secondary metabolites production, we developed a miniaturised screening method which includes decreased cultivation volume, fast extraction and an optimised LC-MS analysis format. Using the UV mutagenesis, we were able to identify several mutants with a higher scopularide production in comparison to the wild type. One of these mutants, which produces three times more biomass and more than double the amount of scopularide A, has been used for another round of mutation. Next generation sequencing is being employed to identify the molecular genetic basis of the observed mutations. In parallel we employ transposable elements to introduce mutants [2]. The impact of transposons on gene expression as well as their ability to cause major mutations within the genome or single genes makes them an interesting tool for random mutagenesis [3, 4, 5]. We employ the Vader transposon in its homologous host and found that Vader mostly integrates within or very close to genes. Thus it appears to be a useful tool for transposon-mediated mutagenesis in A. niger (6). At current we try to enhance its usability by modifying the Vader element

Development and Validation of a Fast and Optimized Screening Method for Enhanced Production of Secondary Metabolites Using the Marine Scopulariopsis brevicaulis Strain LF580 Producing Anti-Cancer Active Scopularide A and B

Natural compounds from marine fungi are an excellent source for the discovery and development of new drug leads. The distinct activity profiles of the two cyclodepsipeptides scopularide A and B against cancer cell lines set their marine producer strain Scopulariopsis brevicaulis LF580 into the focus of the EU project MARINE FUNGI. One of the main goals was the development of a sustainable biotechnological production process for these compounds. The secondary metabolite production of strain LF580 was optimized by random mutagenesis employing UV radiation. For a fast and reliable detection of the intracellular secondary metabolite production level, a miniaturized bioactivity-independent screening method was developed, as the random mutagenesis yielded a large number of mutants to be analysed quantitatively and none of the existing hyphenated bioassay-dependent screening systems could be applied. The method includes decreased cultivation volume, a fast extraction procedure as well as an optimized LC-MS analysis. We show that deviation could be specifically reduced at each step of the process: The measuring deviation during the analysis could be minimized to 5% and technical deviation occurring in the downstream part to 10–15%. Biological variation during the cultivation process still has the major influence on the overall variation. However, the approach led to a 10-fold reduction of time and similar effects on costs and effort compared to standard reference screening methods. The method was applied to screen the UV-mutants library of Scopulariopsis brevicaulis LF580. For validation purposes, the occurring variations in the miniaturized scale were compared to those in the classical Erlenmeyer flask scale. This proof of concept was performed using the wild type strain and 23 randomly selected mutant strains. One specific mutant strain with an enhanced production behavior could be obtained

The Repetitive Landscape of the Barley Genome

While transposable elements (TEs) comprise the bulk of plant genomic DNA, how they contribute to genome structure and organization is still poorly understood. Especially, in large genomes where TEs make the majority of genomic DNA, it is still unclear whether TEs target specific chromosomal regions or whether they simply accumulate where they are best tolerated. The barley genome with its vast repetitive fraction is an ideal system to study chromosomal organization and evolution of TEs. Genes make only about 2% of the genome, while over 80% is derived from TEs. The TE fraction is composed of at least 350 different families. However, 50% of the genome is comprised of only 15 high-copy TE families, while all other TE families are present in moderate or low-copy numbers. The barley genome is highly compartmentalized with different types of TEs occupying different chromosomal “niches”, such as distal, interstitial or proximal regions of chromosome arms. Furthermore, gene space represents its own distinct genomic compartment that is enriched in small non-autonomous DNA transposons, suggesting that these TEs specifically target promoters and downstream regions. Some TE families also show a strong preference to insert in specific sequence motifs which may, in part, explain their distribution. The family-specific distribution patterns result in distinct TE compositions of different chromosomal compartments.Peer reviewe

Genome-Wide Analysis of the “Cut-and-Paste” Transposons of Grapevine

Background: The grapevine is a widely cultivated crop and a high number of different varieties have been selected since its domestication in the Neolithic period. Although sexual crossing has been a major driver of grapevine evolution, its vegetative propagation enhanced the impact of somatic mutations and has been important for grapevine diversity. Transposable elements are known to be major contributors to genome variability and, in particular, to somatic mutations. Thus, transposable elements have probably played a major role in grapevine domestication and evolution. The recent publication of the complete grapevine genome opens the possibility for an in deep analysis of its transposon content. Principal Findings: We present here a detailed analysis of the ‘‘cut-and-paste’ ’ class II transposons present in the genome of grapevine. We characterized 1160 potentially complete grapevine transposons as well as 2086 defective copies. We report on the structure of each element, their potentiality to encode a functional transposase, and the existence of matching ESTs that could suggest their transcription. Conclusions: Our results show that these elements have transduplicated and amplified cellular sequences and some of them have been domesticated and probably fulfill cellular functions. In addition, we provide evidences that the mobility o

<em>Aspergillus nidulans</em> Synthesize Insect Juvenile Hormones upon Expression of a Heterologous Regulatory Protein and in Response to Grazing by <em>Drosophila melanogaster</em> Larvae.

Secondary metabolites are known to serve a wide range of specialized functions including communication, developmental control and defense. Genome sequencing of several fungal model species revealed that the majority of predicted secondary metabolite related genes are silent in laboratory strains, indicating that fungal secondary metabolites remain an underexplored resource of bioactive molecules. In this study, we combine heterologous expression of regulatory proteins in Aspergillus nidulans with systematic variation of growth conditions and observe induced synthesis of insect juvenile hormone-III and methyl farnesoate. Both compounds are sesquiterpenes belonging to the juvenile hormone class. Juvenile hormones regulate developmental and metabolic processes in insects and crustaceans, but have not previously been reported as fungal metabolites. We found that feeding by Drosophila melanogaster larvae induced synthesis of juvenile hormone in A. nidulans indicating a possible role of juvenile hormone biosynthesis in affecting fungal-insect antagonisms

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

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 ∼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

The Oxytricha trifallax Mitochondrial Genome

The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (∼70 kb) plus a ∼5-kb linear plasmid bearing mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps- 2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence. The increased size of the Oxytricha mitochondrial genome relative to other ciliates is primarily a consequence of terminal expansions, rather than the retention of ancestral mitochondrial genes. Successive segmental duplications, visible in one of the two Oxytricha mitochondrial subterminal regions, appear to have contributed to the genome expansion. Consistent with pseudogene formation and decay, the subtermini possess shorter, more loosely packed open reading frames than the remainder of the genome. The mitochondrial plasmid shares a 251-bp region with 82% identity to the mitochondrial chromosome, suggesting that it most likely integrated into the chromosome at least once. This region on the chromosome is also close to the end of the most terminal member of a series of duplications, hinting at a possible association between the plasmid and the duplications. The presence of mitochondrial telomeres on the mitochondrial plasmid suggests that such plasmids may be a vehicle for lateral transfer of telomeric sequences between mitochondrial genomes. We conjecture that the extreme divergence observed in ciliate mitochondrial genomes may be due, in part, to repeated invasions by relatively error-prone DNA polymerase-bearing mobile elements

The methylated component of the Neurospora crassa genome

Cytosine methylation is common, but not ubiquitous, in eukaryotes. Mammals (1) and the fungus Neurospora crassa (2,3) have about 2–3% of cytosines methylated. In mammals, methylation is almost exclusively in the under-represented CpG dinucleotides, and most CpGs are methylated (1) whereas in Neurospora, methylation is not preferentially in CpG dinucleotides and the bulk of the genome is unmethylated (4). DNA methylation is essential in mammals (5) but is dispensable in Neurospora (3,6) making this simple eukaryote a favoured organism in which to study methylation. Recent studies indicate that DNA methylation in Neurospora depends on one DNA methyltransferase, DIM-2 (ref. 6), directed by a histone H3 methyltransferase, DIM-5 (ref. 7), but little is known about its cellular and evolutionary functions. As only four methylated sequences have been reported previously in N. crassa, we used methyl-binding-domain agarose chromatography (8) to isolate the methylated component of the genome. DNA sequence analysis shows that the methylated component of the genome consists almost exclusively of relics of transposons that were subject to repeat-induced point mutation—a genome defence system that mutates duplicated sequences (9)