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During the last two decades, whole-genome sequencing has revolutionized genetic research in all kingdoms, including fungi. More than 1000 fungal genomes have been submitted to sequence databases, mostly obtained through second generation short-read DNA sequencing. As a result, highly fragmented genome drafts have typically been obtained. However, with the emergence of third generation long-read DNA sequencing, the assembly challenge can be overcome and highly contiguous assemblies obtained. Such attractive results, however, are extremely dependent on the ability to extract highly purified high molecular weight (HMW) DNA. Extraction of such DNA is currently a significant challenge for all species with cell walls, not least fungi. In this study, four isolates of filamentous ascomycetes (Apiospora pterospermum, Aspergillus sp. (subgen. Cremei), Aspergillus westerdijkiae, and Penicillium aurantiogriseum) were used to develop extraction and purification methods that result in HMW DNA suitable for third generation sequencing. We have tested and propose two straightforward extraction methods based on treatment with either a commercial kit or traditional phenol-chloroform extraction both in combination with a single commercial purification method that result in high quality HMW DNA from filamentous ascomycetes. Our results demonstrated that using these DNA extraction methods and coverage, above 75 x of our haploid filamentous ascomycete fungal genomes result in complete and contiguous assemblies.

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.

The antimicrobial activity of the phenothiazine derivatives thioridazine and prochlorperazine have been evaluated with 11 Enterococcus faecalis strains and 9 Enterococcus faecium strains, originating from human infections and animal faecal flora. We found that all E. faecalis and E. faecium strains, regardless of their susceptibility to commonly used antibiotics, were inhibited by thioridazine at a concentration of 16-32 microg/ml and by prochlorperazine at a concentration of 32-64 microg/ml. Combinations of the antibiotics vancomycin or ampicillin and thioridazine and prochlorperazine at subinhibitory concentrations, could render vancomycin- or ampicillin-resistant bacteria sensitive to each of the antibiotics. Verapamil and reserpine, inhibitors of P-glycoprotein-mediated multidrug resistance, did not reduce resistance. Our results outline modification of resistance in enterococci induced by phenothiazine derivatives unrelated to P-glycoprotein-mediated multidrug resistance.

The anti-microbial activity of six non-antibiotics (one amino-ethylchloride, three phenothiazines, two tricyclic antidepressives) were tested on 20 clinical isolates of Pseudomonas aeruginosa , one clinical isolate of Klebsiella pneumoniae , 2 ATTC strains and 14 clinical isolates of Staphylococccus aureus , using the plate dilution method. The effects on P. aeruginosa were independent of antibiotic resistance pattern and the species Stenotrophomonas maltophilia was found to be the most susceptible to the non-antibiotics, with MIC values as low as 20 mg/l for some of the substances. The 16 S. aureus strains tested were all particularly susceptible to the anti-microbial effects of the putative inhibitors of efflux pumps thioridazine and trifluoperazine with MIC values of ≤16 mg/l independently of the methicillin resistance profile of the strains. Because phenothiazines are well known to inhibit efflux pumps our results may indicate the existence of such pumps. Current works in progress are attempts at reversing the antibiotic resistance of selected bacterial strains using specific non-antibiotics and their stereo-chemical isomers.

Through stepwise recreation of the biosynthetic gene cluster containing PKS3 from Fusarium solani, it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in Saccharomyces cerevisiae. This was achieved through sequential transformation associated recombination (TAR) cloning of FvPPT, fsr1, fsr2, and fsr3 into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in S. cerevisiae. The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1–4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L.