The Penicillia are known to produce a wide range natural products—some with devastating outcome for the agricultural industry and others with unexploited potential in different applications. However, a large-scale overview of the biosynthetic potential of different species has been lacking. In this study, we sequenced 93 Penicillium isolates and, together with eleven published genomes that hold similar assembly characteristics, we established a species phylogeny as well as defining a Penicillium pangenome. A total of 5612 genes were shared between ≥ 98 isolates corresponding to approximately half of the average number of genes a Penicillium genome holds. We further identified 15 lateral gene transfer events that have occurred in this collection of Penicillium isolates, which might have played an important role, such as niche adaption, in the evolution of these fungi. The comprehensive characterization of the genomic diversity in the Penicillium genus supersedes single-reference genomes, which do not necessarily capture the entire genetic variation.

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.

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.