Project description:The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.
Project description:Using transcriptomics, the strain-specific metabolism was mapped for two whole-genome sequenced strains of Aspergillus niger Keywords: Strain comparison
Project description:Using transcriptomics, the strain-specific metabolism was mapped for two whole-genome sequenced strains of Aspergillus niger Keywords: Strain comparison Two strains grown in controlled bioreactors, three biological replicates each.
Project description:Fungal infections pose a great threat to public health and the existing four classes of antifungals have limitations due to high toxicity, drug-drug interactions, and emerging drug-resistance. Streptomyces spp. represent an important source of antimicrobial substances, notably including the antifungal agent amphotericin B. The rapamycin-producer Streptomyces iranensis displayed strong antifungal activities against Aspergillus. Revisiting its genome revealed several intriguing biosynthetic gene clusters, including one unparalleled Type I polyketide synthase, which codes for uncharacterized metabolites. The identification of a novel macrolide spirolactone was facilitated through CRISPR-based gene editing, HR-ESI-MS analysis, followed by fermentation and purification processes. Their structures and absolute configurations were confirmed by NMR, MS and X-ray crystallography. Spirolactone A harbors an undescribed carbon skeleton with 13 chiral centers, featuring a rare ?-lactone moiety, a [6,6]-spiroketal ring, and an unprecedented 7-oxo-octylmalonyl-CoA extender unit. Spirolactone displayed profound antifungal effects against numerous fungal pathogens, e.g. the genus Talaromyces and several sections of Aspergillus including clinically relevant species such as Aspergillus niger and A. tubingensis (section Nigri), A. terreus (section Terrei) and the azol-resistant A. calidoustus (section Usti). Proteomics analysis revealed spirolactone potentially disrupted the integrity of fungal cell walls and induced the expression of stress-response proteins in A. niger. Spirolactone A represents a new class of potential agents leading to combat fungal infections.
Project description:Azaphilones are a class of fungal metabolites characterized by a highly oxygenated pyrano-quinone bicyclic core and exhibiting a broad range of bioactivities. Although widespread among various fungi, their biosynthesis has not been thoroughly elucidated. By activation of a silent (aza) gene cluster in Aspergillus niger ATCC 1015, we discovered six azaphilone compounds, azanigerones A-F (1, 3-7). Transcriptional analysis and deletion of a key polyketide synthase (PKS) gene further confirmed the involvement of the aza gene cluster. The biosynthetic pathway was shown to involve the convergent actions of a highly reducing PKS and a non-reducing PKS. Most significantly, in vitro reaction of a key flavin-dependent monooxygenase encoded in the cluster with an early benzaldehyde intermediate revealed its roles in hydroxylation and pyran-ring formation to afford the characteristic bicylic core shared by azaphilones.
Project description:Aspergillus niger is well known for its capability to produce citrate in high amounts but the detailed metabolic response causing citrate production has not been fully elucidated. Manganese is known to have an important effect as its limitation is a requirement to obtain high-level citrate formation. To identify the translational regulation causing citric acid overflow metabolism, transcriptome and proteome data from cultivations in manganese limitation and manganese excess conditions were analyzed. In addition to four already described main responses, two novel events were identified. The first metabolic response was down regulation of phosphoenolpyruvate carboxykinase (PEPCK) during manganese limited conditions, which was confirmed by in vivo experiments. Down regulation of the first step in the gluconeogenesis, while maintaining a high activity through glycolysis, promoted secretion of citrate into the medium as an alternative regulatory mechanism for adjusting the intracellular concentrations of TCA intermediates. The other novel observation was down regulation of two cation transporters at manganese limited conditions. It was hypothesized that lowered cation transport across the mitochondrial membrane reduced the ability of the cell to maintain homeostasis thereby favoring citric acid secretion. Finally, upregulation of an ABC transporter was measured, which was assumed to be a citrate permease. 9 samples in total. Three conditions in triplicates
Project description:Aspergillus niger is well known for its capability to produce citrate in high amounts but the detailed metabolic response causing citrate production has not been fully elucidated. Manganese is known to have an important effect as its limitation is a requirement to obtain high-level citrate formation. To identify the translational regulation causing citric acid overflow metabolism, transcriptome and proteome data from cultivations in manganese limitation and manganese excess conditions were analyzed. In addition to four already described main responses, two novel events were identified. The first metabolic response was down regulation of phosphoenolpyruvate carboxykinase (PEPCK) during manganese limited conditions, which was confirmed by in vivo experiments. Down regulation of the first step in the gluconeogenesis, while maintaining a high activity through glycolysis, promoted secretion of citrate into the medium as an alternative regulatory mechanism for adjusting the intracellular concentrations of TCA intermediates. The other novel observation was down regulation of two cation transporters at manganese limited conditions. It was hypothesized that lowered cation transport across the mitochondrial membrane reduced the ability of the cell to maintain homeostasis thereby favoring citric acid secretion. Finally, upregulation of an ABC transporter was measured, which was assumed to be a citrate permease.