Penicillium roqueforti is 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... Show morePenicillium roqueforti is 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 (MICu) ranges between 4.2 and 21.2 mM when 34 P. roqueforti strains 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 sensitive P. roqueforti strains that were differentially expressed in the presence of the weak acid. These genes could explain why P. roqueforti is more resistant to sorbic acid when compared to other fungi, even in the absence of the SORBUS cluster. Together, the MICu of 21.2 mM makes P. roqueforti among the most sorbic acid-resistant fungi, if not the most resistant fungus, which is mediated by the SORBUS gene cluster. Show less
Mating-type distribution within a phylogenetic tree, heterokaryon compatibility, and subsequent diploid formation were studied in 24 Aspergillus niger sensu stricto strains. The genomes of the 24... Show moreMating-type distribution within a phylogenetic tree, heterokaryon compatibility, and subsequent diploid formation were studied in 24 Aspergillus niger sensu stricto strains. The genomes of the 24 strains were sequenced and analyzed revealing an average of 6.1 ± 2.0 variants/kb between Aspergillus niger sensu stricto strains. The genome sequences were used together with available genome data to generate a phylogenetic tree revealing 3 distinct clades within Aspergillus niger sensu stricto. The phylogenetic tree revealed that both MAT1-1 and MAT1-2 mating types were present in each of the 3 clades. The phylogenetic differences were used to select for strains to analyze heterokaryon compatibility. Conidial color markers (fwnA and brnA) and auxotrophic markers (pyrG and nicB) were introduced via CRISPR/Cas9-based genome editing in a selection of strains. Twenty-three parasexual crosses using 11 different strains were performed. Only a single parasexual cross between genetically highly similar strains resulted in a successful formation of heterokaryotic mycelium and subsequent diploid formation, indicating widespread heterokaryon incompatibility as well as multiple active heterokaryon incompatibility systems between Aspergillus niger sensu stricto strains. The 2 vegetatively compatible strains were of 2 different mating types and a stable diploid was isolated from this heterokaryon. Sclerotium formation was induced on agar media containing Triton X-100; however, the sclerotia remained sterile and no ascospores were observed. Nevertheless, this is the first report of a diploid Aspergillus niger sensu stricto strain with 2 different mating types, which offers the unique possibility to screen for conditions that might lead to ascospore formation in A. niger.Show less
Brule, T. van den; Punt, M.; Seekles, S.J.; Segers, F.J.J.; Houbraken, J.; Hazeleger, W.C.; ... ; Besten, H.M.W. den 2022
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... Show moreMicrobial 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. Show less
Seekles, S.J.; Dam, J. van; Arentshorst, M.; Ram, A.F.J. 2022
Weak acids, such as sorbic acid, are used as chemical food preservatives by the industry. Fungiovercome this weak-acid stress by inducing cellular responses mediated by transcription factors. In... Show moreWeak acids, such as sorbic acid, are used as chemical food preservatives by the industry. Fungiovercome this weak-acid stress by inducing cellular responses mediated by transcription factors. In ourresearch, a large-scale sorbic acid resistance screening was performed on 100 A. niger sensu stricto strains isolated fromvarious sources to study strain variability in sorbic acid resistance. Theminimal inhibitory concentration of undissociated (MICu) sorbic acid at pH = 4 in the MEB of the A. niger strains varies between 4.0 mMand 7.0 mM, with the average out of 100 strains being 4.8 0.8 mM, when scored after 28 days. MICu valueswere roughly 1mMlowerwhen tested in commercial ice tea. Genome sequencingof the most sorbic-acid-sensitive strain among the isolates revealed a premature stop codon inside thesorbic acid response regulator encoding gene sdrA. Repairing this missense mutation increased thesorbic acid resistance, showing that the sorbic-acid-sensitive phenotype of this strain is caused by theloss of SdrA function. To identify additional transcription factors involved in weak-acid resistance,a transcription factor knock-out library consisting of 240 A. niger deletion strains was screened. Thescreen identified a novel transcription factor,WarB, which contributes to the resistance against a broadrange of weak acids, including sorbic acid. The roles of SdrA,WarA andWarB in weak-acid resistance,including sorbic acid, were compared by creating single, double and the triple knock-out strains. Allthree transcription factors were found to have an additive effect on the sorbic acid stress response. Show less
Fungal food spoilage often starts with a contamination with spores. Experimental data strongly indicate the existence of subpopulations of spores with different levels of resistance to preservation... Show moreFungal food spoilage often starts with a contamination with spores. Experimental data strongly indicate the existence of subpopulations of spores with different levels of resistance to preservation methods. In this thesis, the extent of this heterogeneity and the underlying mechanisms using fungal model systems is studied. The role of the genetic background, environmental conditions and the developmental state of the spores were studied, using quantitative imaging, genome and RNA/protein sequencing as well as functional gene analysis. The role of transcription factors in weak acid stress resistance of Aspergillus niger is described. Next, heat resistance of fungal spores of three food spoilage species was quantified and compared. The genomes of Aspergillus niger strains were sequenced and compared revealing the existence of a possible sexual cycle. Melanin of fungal spores impacts UV-C resistance, but not heat resistance and a functional CRISPR/Cas9 genome editing system for Paecilomyces variotii and Penicillium roqueforti is described. Older spores are more heat resistant than younger spores, which can be contributed to differences in compatible solute composition. Additionally, a high cultivation temperature results in fungal spores with high heat resistance, possibly due to heat shock proteins. Show less
BackgroundAspergillus niger is a ubiquitous filamentous fungus widely employed as a cell factory thanks to its abilities to produce a wide range of organic acids and enzymes. Its genome was one of... Show moreBackgroundAspergillus niger is a ubiquitous filamentous fungus widely employed as a cell factory thanks to its abilities to produce a wide range of organic acids and enzymes. Its genome was one of the first Aspergillus genomes to be sequenced in 2007, due to its economic importance and its role as model organism to study fungal fermentation. Nowadays, the genome sequences of more than 20 A. niger strains are available. These, however, do not include the neotype strain CBS 554.65.ResultsThe genome of CBS 554.65 was sequenced with PacBio. A high-quality nuclear genome sequence consisting of 17 contigs with a N50 value of 4.07 Mbp was obtained. The assembly covered all the 8 centromeric regions of the chromosomes. In addition, a complete circular mitochondrial DNA assembly was obtained. Bioinformatic analyses revealed the presence of a MAT1-2-1 gene in this genome, contrary to the most commonly used A. niger strains, such as ATCC 1015 and CBS 513.88, which contain a MAT1-1-1 gene. A nucleotide alignment showed a different orientation of the MAT1–1 locus of ATCC 1015 compared to the MAT1–2 locus of CBS 554.65, relative to conserved genes flanking the MAT locus. Within 24 newly sequenced isolates of A. niger half of them had a MAT1–1 locus and the other half a MAT1–2 locus. The genomic organization of the MAT1–2 locus in CBS 554.65 is similar to other Aspergillus species. In contrast, the region comprising the MAT1–1 locus is flipped in all sequenced strains of A. niger.ConclusionsThis study, besides providing a high-quality genome sequence of an important A. niger strain, suggests the occurrence of genetic flipping or switching events at the MAT1–1 locus of A. niger. These results provide new insights in the mating system of A. niger and could contribute to the investigation and potential discovery of sexuality in this species long thought to be asexual. Show less
Seekles, S.J.; Teunisse, P. P. P.; Punt M.; Brule, T. van den; Dijksterhuis, J.; Houbraken, J.; ... ; Ram, A.F.J. 2021
BackgroundThe 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... Show moreBackgroundThe 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.ResultsHere 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.ConclusionsOur 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. Show less
