Background: The filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti. Results: Here we describe the successful implementation of an efficient AMA1-based CRISPR/Cas9 genome editing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin deficient strains. Additionally, kusA⁻ mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efficient genome editing in these species. The effect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin deficient mutant conidia. Conclusions: Our findings show the successful use of CRISPR/Cas9 genome editing and its high efficiency in P. variotii and P. roqueforti in both wild-type strains as well as kusA⁻ mutant background strains. Additionally, we observed that melanin deficient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin deficient conidia had increased sensitivity towards UV-C radiation.

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9‐mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9‐compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 μg·mL⁻¹ PatE protein in the culture medium with a purity just under 90%.

Background: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle. Results: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance. Conclusions: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Microbial species are inherently variable, which is reflected in intraspecies genotypic and phenotypic differences. Strain-to-strain variation gives rise to variability in stress resistance and plays a crucial role in food safety and food quality. Here, strain variability in heat resistance of asexual spores (conidia) of the fungal species Aspergillus niger, Penicillium roqueforti and Paecilomyces variotii was quantified and compared to bacterial variability found in the literature. After heat treatment, a 5.4- to 8.6-fold difference in inactivation rate was found between individual strains within each species, while the strain variability of the three fungal species was not statistically different. We evaluated whether the degree of intraspecies variability is uniform, not only within the fungal kingdom, but also amongst different bacterial species. Comparison with three spore-forming bacteria and two non-spore-forming bacteria revealed that the variability of the different species was indeed in the same order of magnitude, which hints to a microbial signature of variation that exceeds kingdom boundaries.

Penicillium roquefortiis a major food-spoilage fungus known for its high resistance to the food preservative sorbic acid. Here, we demonstrate that the minimum inhibitory concentration of undissociated sorbic acid (MIC_u) ranges between 4.2 and 21.2 mM when 34P.roquefortistrains were grown on malt extract broth. A genome-wide association study revealed that the six most resistant strains contained the 180 kbp gene cluster SORBUS, which was absent in the other 28 strains. In addition, a SNP analysis revealed five genes outside the SORBUS cluster that may be linked to sorbic acid resistance. A partial SORBUS knock-out (>100 of 180 kbp) in a resistant strain reduced sorbic acid resistance to similar levels as observed in the sensitive strains. Whole genome transcriptome analysis revealed a small set of genes present in both resistant and sensitiveP.roquefortistrains that were differentially expressed in the presence of the weak acid. These genes could explain whyP.roquefortiis more resistant to sorbic acid when compared to other fungi, even in the absence of the SORBUS cluster. Together, the MIC_uof 21.2 mM makesP.roquefortiamong the most sorbic acid-resistant fungi, if not the most resistant fungus, which is mediated by the SORBUS gene cluster.

The transition from dormant spore to germling has been topic of study and debate. A recent discovery in PLOS Biology shows that chaperone Hsp42 plays a crucial role in resolubilizing the proteome during dormancy breaking, although a role of trehalose cannot be excluded.