Protoplast-mediated transformation of Madurella mycetomatis using hygromycin resistance as a selection marker.

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies.

Compatible solutes determine the heat resistance of conidia.

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.

Functional analysis of the protocatechuate branch of the ß-ketoadipate pathway in Aspergillus niger.

Bacteria and fungi catabolize plant-derived aromatic compounds by funneling into one of seven dihydroxylated aromatic intermediates, which then undergo ring fission and conversion to TCA cycle intermediates. Two of these intermediates, protocatechuic acid and catechol, converge on ß-ketoadipate which is further cleaved to succinyl-CoA and acetyl-CoA. These ß-ketoadipate pathways have been well characterized in bacteria. The corresponding knowledge of these pathways in fungi is incomplete. Characterization of these pathways in fungi would expand our knowledge and improve the valorization of lignin-derived compounds. Here, we used homology to characterize bacterial or fungal genes to predict the genes involved in the ß-ketoadipate pathway for protocatechuate utilization in the filamentous fungus Aspergillus niger. We further used the following approaches to refine the assignment of the pathway genes: whole transcriptome sequencing to reveal genes upregulated in the presence of protocatechuic acid; deletion of candidate genes to observe their ability to grow on protocatechuic acid; determination by mass spectrometry of metabolites accumulated by deletion mutants; and enzyme assays of the recombinant proteins encoded by candidate genes. Based on the aggregate experimental evidence, we assigned the genes for the five pathway enzymes as follows: NRRL3_01405 (prcA) encodes protocatechuate 3,4-dioxygenase; NRRL3_02586 (cmcA) encodes 3-carboxy-cis,cis-muconate cyclase; NRRL3_01409 (chdA) encodes 3-carboxymuconolactone hydrolase/decarboxylase; NRRL3_01886 (kstA) encodes ß-ketoadipate:succinyl-CoA transferase; and NRRL3_01526 (kctA) encodes ß-ketoadipyl-CoA thiolase. Strain carrying ΔNRRL3_00837 could not grow on protocatechuic acid, suggesting that it is essential for protocatechuate catabolism. Its function is unknown as recombinant NRRL3_00837 did not affect the in vitro conversion of protocatechuic acid to ß-ketoadipate.

A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger.

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%.

Structure elucidation and characterization of patulin synthase, insights into the formation of a fungal mycotoxin.

Patulin synthase (PatE) from Penicillium expansum is a flavin-dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in fruit and fruit-derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. This confirmed that PatE is active not only on the proposed patulin precursor ascladiol but also on several aromatic alcohols including 5-hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl-alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin.

Natural Variation and the Role of Zn2Cys6 Transcription Factors SdrA, WarA and WarB in Sorbic Acid Resistance of Aspergillus niger.

Weak acids, such as sorbic acid, are used as chemical food preservatives by the industry. Fungi overcome this weak-acid stress by inducing cellular responses mediated by transcription factors. In our research, a large-scale sorbic acid resistance screening was performed on 100 A. niger sensu stricto strains isolated from various sources to study strain variability in sorbic acid resistance. The minimal inhibitory concentration of undissociated (MICu) sorbic acid at pH = 4 in the MEB of the A. niger strains varies between 4.0 mM and 7.0 mM, with the average out of 100 strains being 4.8 ± 0.8 mM, when scored after 28 days. MICu values were roughly 1 mM lower when tested in commercial ice tea. Genome sequencing of the most sorbic-acid-sensitive strain among the isolates revealed a premature stop codon inside the sorbic acid response regulator encoding gene sdrA. Repairing this missense mutation increased the sorbic acid resistance, showing that the sorbic-acid-sensitive phenotype of this strain is caused by the loss 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. The screen identified a novel transcription factor, WarB, which contributes to the resistance against a broad range of weak acids, including sorbic acid. The roles of SdrA, WarA and WarB in weak-acid resistance, including sorbic acid, were compared by creating single, double and the triple knock-out strains. All three transcription factors were found to have an additive effect on the sorbic acid stress response.

Utilization of ferulic acid in Aspergillus niger requires the transcription factor FarA and a newly identified Far-like protein (FarD) that lacks the canonical Zn(II)2Cys6 domain.

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux613 dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)2Cys6 domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)2Cys6 domain and possesses only the fungal specific transcription factor domain.

Genetic Characterization of Mutations Related to Conidiophore Stalk Length Development in Aspergillus niger Laboratory Strain N402.

Aspergillus niger is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the A. niger N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of A. niger, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of A. niger strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed cspA and cspB) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (cspA) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the A. nidulans UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (cspB) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.

Loss of function of the carbon catabolite repressor CreA leads to low but inducer-independent expression from the feruloyl esterase B promoter in Aspergillus niger.

OBJECTIVE: With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter. RESULT: PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter. CONCLUSION: Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

Identification of a Conserved Transcriptional Activator-Repressor Module Controlling the Expression of Genes Involved in Tannic Acid Degradation and Gallic Acid Utilization in Aspergillus niger.

Tannic acid, a hydrolysable gallotannin present in plant tissues, consists of a central glucose molecule esterified with gallic acid molecules. Some microorganisms, including several Aspergillus species, can metabolize tannic acid by releasing gallic acid residues from tannic acid by secreting tannic acid specific esterases into the medium. The expression of these so-called tannases is induced by tannic acid or gallic acid. In this study, we identified a conserved transcriptional activator-repressor module involved in the regulation of predicted tannases and other genes involved in gallic acid metabolism. The transcriptional activator-repressor module regulating tannic acid utilization resembles the transcriptional activator-repressor modules regulating galacturonic acid and quinic acid utilization. Like these modules, the Zn(II)2Cys6 transcriptional activator (TanR) and the putative repressor (TanX) are located adjacent to each other. Deletion of the transcriptional activator (ΔtanR) results in inability to grow on gallic acid and severely reduces growth on tannic acid. Deletion of the putative repressor gene (ΔtanX) results in the constitutive expression of tannases as well as other genes with mostly unknown function. Known microbial catabolic pathways for gallic acid utilization involve so-called ring cleavage enzymes, and two of these ring cleavage enzymes show increased expression in the ΔtanX mutant. However, deletion of these two genes, and even deletion of all 17 genes encoding potential ring cleavage enzymes, did not result in a gallic acid non-utilizing phenotype. Therefore, in A. niger gallic acid utilization involves a hitherto unknown pathway. Transcriptome analysis of the ΔtanX mutant identified several genes and gene clusters that were significantly induced compared to the parental strain. The involvement of a selection of these genes and gene clusters in gallic acid utilization was examined by constructing gene deletion mutants and testing their ability to grow on gallic acid. Only the deletion of a gene encoding an FAD-dependent monooxygenase (NRRL3_04659) resulted in a strain that was unable to grow on gallic acid. Metabolomic studies showed accumulation of gallic acid in the ΔNRRL3_04659 mutant suggesting that this predicted monooxygenase is involved in the first step of gallic acid metabolism and is likely responsible for oxidation of the aromatic ring.

Deletion of the Aspergillus niger Pro-Protein Processing Protease Gene kexB Results in a pH-Dependent Morphological Transition during Submerged Cultivations and Increases Cell Wall Chitin Content.

There is a growing interest in the use of post-fermentation mycelial waste to obtain cell wall chitin as an added-value product. In the pursuit to identify suitable production strains that can be used for post-fermentation cell wall harvesting, we turned to an Aspergillus niger strain in which the kexB gene was deleted. Previous work has shown that the deletion of kexB causes hyper-branching and thicker cell walls, traits that may be beneficial for the reduction in fermentation viscosity and lysis. Hyper-branching of ∆kexB was previously found to be pH-dependent on solid medium at pH 6.0, but was absent at pH 5.0. This phenotype was reported to be less pronounced during submerged growth. Here, we show a series of controlled batch cultivations at a pH range of 5, 5.5, and 6 to examine the pellet phenotype of ΔkexB in liquid medium. Morphological analysis showed that ΔkexB formed wild type-like pellets at pH 5.0, whereas the hyper-branching ΔkexB phenotype was found at pH 6.0. The transition of phenotypic plasticity was found in cultivations at pH 5.5, seen as an intermediate phenotype. Analyzing the cell walls of ΔkexB from these controlled pH-conditions showed an increase in chitin content compared to the wild type across all three pH values. Surprisingly, the increase in chitin content was found to be irrespective of the hyper-branching morphology. Evidence for alterations in cell wall make-up are corroborated by transcriptional analysis that showed a significant cell wall stress response in addition to the upregulation of genes encoding other unrelated cell wall biosynthetic genes.

A seven-membered cell wall related transglycosylase gene family in Aspergillus niger is relevant for cell wall integrity in cell wall mutants with reduced α-glucan or galactomannan.

Chitin is an important fungal cell wall component that is cross-linked to ß-glucan for structural integrity. Acquisition of chitin to glucan cross-links has previously been shown to be performed by transglycosylation enzymes in Saccharomyces cerevisiae, called Congo Red hypersensitive (Crh) enzymes. Here, we characterized the impact of deleting all seven members of the crh gene family (crhA-G) in Aspergillus niger on cell wall integrity, cell wall composition and genome-wide gene expression. In this study, we show that the seven-fold crh knockout strain shows slightly compact growth on plates, but no increased sensitivity to cell wall perturbing compounds. Additionally, we found that the cell wall composition of this knockout strain was virtually identical to that of the wild type. In congruence with these data, genome-wide expression analysis revealed very limited changes in gene expression and no signs of activation of the cell wall integrity response pathway. However, deleting the entire crh gene family in cell wall mutants that are deficient in either galactofuranose or α-glucan, mainly α-1,3-glucan, resulted in a synthetic growth defect and an increased sensitivity towards Congo Red compared to the parental strains, respectively. Altogether, these results indicate that loss of the crh gene family in A. niger does not trigger the cell wall integrity response, but does play an important role in ensuring cell wall integrity in mutant strains with reduced galactofuranose or α-glucan.

Interrogation of the cell wall integrity pathway in Aspergillus niger identifies a putative negative regulator of transcription involved in chitin deposition.

Post-fermentation fungal biomass waste provides a viable source for chitin. Cell wall chitin of filamentous fungi, and in particular its de-N-acetylated derivative chitosan, has a wide range of commercial applications. Although the cell wall of filamentous fungi comprises 10-30% chitin, these yields are too low for cost-effective production. Therefore, we aimed to identify the genes involved in increased chitin deposition by screening a collection of UV-derived cell wall mutants in Aspergillus niger. This screen revealed a mutant strain (RD15.4#55) that showed a 30-40% increase in cell wall chitin compared to the wild type. In addition to the cell wall chitin phenotype, this strain also exhibited sensitivity to SDS and produces an unknown yellow pigment. Genome sequencing combined with classical genetic linkage analysis identified two mutated genes on chromosome VII that were linked with the mutant phenotype. Single gene knockouts and subsequent complementation analysis revealed that an 8 bp deletion in NRRL3_09595 is solely responsible for the associated phenotypes of RD15.4#55. The mutated gene, which was named cwcA (cell wall chitin A), encodes an orthologue of Saccharomyces cerevisiae Bypass of ESS1 (BYE1), a negative regulator of transcription elongation. We propose that this conserved fungal protein is involved in preventing cell wall integrity signaling under non-inducing conditions, where loss of function results in constitutive activation of the cell wall stress response pathway, and consequently leads to increased chitin content in the mutant cell wall.

Identification of SclB, a Zn(II)2Cys6 transcription factor involved in sclerotium formation in Aspergillus niger.

Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.

Interrogation of the cell wall integrity pathway in Aspergillus niger identifies a putative negative regulator of transcription involved in chitin deposition.

Post-fermentation fungal biomass waste provides a viable source for chitin. Cell wall chitin of filamentous fungi, and in particular its de-N-acetylated derivative chitosan, has a wide range of commercial applications. Although the cell wall of filamentous fungi comprises 10-30% chitin, these yields are too low for cost-effective production. Therefore, we aimed to identify the genes involved in increased chitin deposition by screening a collection of UV-derived cell wall mutants in Aspergillus niger. This screen revealed a mutant strain (RD15.4#55) that showed a 30-40% increase in cell wall chitin compared to the wild type. In addition to the cell wall chitin phenotype, this strain also exhibited sensitivity to SDS and produces an unknown yellow pigment. Genome sequencing combined with classical genetic linkage analysis identified two mutated genes on chromosome VII that were linked with the mutant phenotype. Single gene knockouts and subsequent complementation analysis revealed that an 8 bp deletion in NRRL3_09595 is solely responsible for the associated phenotypes of RD15.4#55. The mutated gene, which was named cwcA (cell wall chitin A), encodes an orthologue of Saccharomyces cerevisiae Bypass of ESS1 (BYE1), a negative regulator of transcription elongation. We propose that this conserved fungal protein is involved in preventing cell wall integrity signaling under non-inducing conditions, where loss of function results in constitutive activation of the cell wall stress response pathway, and consequently leads to increased chitin content in the mutant cell wall.

Functional analysis of three putative galactofuranosyltransferases with redundant functions in galactofuranosylation in Aspergillus niger.

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.

Rab GDP-dissociation inhibitor gdiA is an essential gene required for cell wall chitin deposition in Aspergillus niger.

The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5' donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.

Efficient marker free CRISPR/Cas9 genome editing for functional analysis of gene families in filamentous fungi.

BACKGROUND: CRISPR/Cas9 mediated genome editing has expedited the way of constructing multiple gene alterations in filamentous fungi, whereas traditional methods are time-consuming and can be of mutagenic nature. These developments allow the study of large gene families that contain putatively redundant genes, such as the seven-membered family of crh-genes encoding putative glucan-chitin crosslinking enzymes involved in cell wall biosynthesis. RESULTS: Here, we present a CRISPR/Cas9 system for Aspergillus niger using a non-integrative plasmid, containing a selection marker, a Cas9 and a sgRNA expression cassette. Combined with selection marker free knockout repair DNA fragments, a set of the seven single knockout strains was obtained through homology directed repair (HDR) with an average efficiency of 90%. Cas9-sgRNA plasmids could effectively be cured by removing selection pressure, allowing the use of the same selection marker in successive transformations. Moreover, we show that either two or even three separate Cas9-sgRNA plasmids combined with marker-free knockout repair DNA fragments can be used in a single transformation to obtain double or triple knockouts with 89% and 38% efficiency, respectively. By employing this technique, a seven-membered crh-gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker (pyrG) recycling transformations. An additional advantage of the use of marker-free gene editing is that negative effects of selection marker gene expression are evaded, as we observed in the case of disrupting virtually silent crh family members. CONCLUSIONS: Our findings advocate the use of CRISPR/Cas9 to create multiple gene deletions in both a fast and reliable way, while simultaneously omitting possible locus-dependent-side-effects of poor auxotrophic marker expression.

Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based Probes.

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of ß-xylosidases and endo-ß-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the ß-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.

Correction to: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.

The correct title is: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.