Antifungal Activity and Biosynthetic Potential of New Streptomyces sp. MW-W600-10 Strain Isolated from Coal Mine Water.

Crop infections by fungi lead to severe losses in food production and pose risks for human health. The increasing resistance of pathogens to fungicides has led to the higher usage of these chemicals, which burdens the environment and highlights the need to find novel natural biocontrol agents. Members of the genus Streptomyces are known to produce a plethora of bioactive compounds. Recently, researchers have turned to extreme and previously unexplored niches in the search for new strains with antimicrobial activities. One such niche are underground coal mine environments. We isolated the new Streptomyces sp. MW-W600-10 strain from coal mine water samples collected at 665 m below ground level. We examined the antifungal activity of the strain against plant pathogens Fusarium culmorum DSM62188 and Nigrospora oryzae roseF7. Furthermore, we analyzed the strain's biosynthetic potential with the antiSMASH tool. The strain showed inhibitory activity against both fungi strains. Genome mining revealed that it has 39 BGCs, among which 13 did not show similarity to those in databases. Additionally, we examined the activity of the Streptomyces sp. S-2 strain isolated from black soot against F. culmorum DSM62188. These results show that coal-related strains could be a source of novel bioactive compounds. Future studies will elucidate their full biotechnological potential.

Identification of the biosynthetic gene clusters for the lipopeptides fusaristatin A and W493 B in Fusarium graminearum and F. pseudograminearum.

The closely related species Fusarium graminearum and Fusarium pseudograminearum differ in that each contains a gene cluster with a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) that is not present in the other species. To identify their products, we deleted PKS6 and NRPS7 in F. graminearum and NRPS32 in F. pseudograminearum. By comparing the secondary metabolite profiles of the strains we identified the resulting product in F. graminearum as fusaristatin A, and as W493 A and B in F. pseudograminearum. These lipopeptides have previously been isolated from unidentified Fusarium species. On the basis of genes in the putative gene clusters we propose a model for biosynthesis where the polyketide product is shuttled to the NPRS via a CoA ligase and a thioesterase in F. pseudograminearum. In F. graminearum the polyketide is proposed to be directly assimilated by the NRPS.

Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum.

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

Genome Sequencing and analyses of Two Marine Fungi from the North Sea Unraveled a Plethora of Novel Biosynthetic Gene Clusters.

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.

Overexpression of NRPS4 leads to increased surface hydrophobicity in Fusarium graminearum.

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.