β-Mannanases are endo-acting glycoside hydrolases (GHs) that cleave β-1,4 glycosidic linkages in mannan-rich plant cell wall polysaccharides. They find application in the food and paper industries. Activity-based probes (ABPs) are powerful tools for GH profiling in complex biological samples, yet to date, bespoke ABPs reporting on mannanases have not been reported. Here, we describe the synthesis of cyclophellitol-inspired ABPs based on mannobiose, mannotriose, and glucomannose, and their use in reporting mannanase activities in secretomes from saprophytic bacteria and fungi grown on mannan-containing biomass polysaccharides. In addition to mannanases, our ABPs also labelled cellulases in secretomes from both Aspergillus niger and Cellvibrio japonicus, which may indicate broader ("negative-subsite") substrate specificity in these enzymes. Mechanistic proof of active-site nucleophile labelling by our ABPs was obtained for both AnManA and CjMan26C by X-ray crystallography and for both AnManA and AnMan26A by mass spectrometry. Together, our results establish mannanase-targeted ABPs that may find use alongside existing reagents that report on retaining GHs that process other bulk polysaccharides.

In this protocol, we describe a robust luciferase-based biosensor assay to monitor the activity of the cell wall integrity (CWI) pathway in Aspergillus fumigatus in real time. The method relies on the stable integration of a markerless, synthetic reporter cassette (p)agsA::luc at the aft4 Safe Haven (Sh) genomic locus using CRISPR-Cas9. This cassette comprises a modified A. niger agsA promoter containing three tandem RlmA-binding sites, which drives the expression of the luciferase gene. Upon exposure to cell wall stress, the endogenous transcription factor RlmA activates the reporter, generating a luminescent signal proportional to promoter activity. The protocol includes the construction of aft4 locus-specific CRISPR-Cas9 plasmids, A. fumigatus transformation and candidate selection, as well as the setup of the luminescence bioassay in white 96-well microplates. This system enables highly sensitive, nondestructive, and time-resolved quantification of CWI pathway activation during early fungal growth or biofilm under various genetic or chemical perturbations. Moreover, it supports comparative studies across wild-type and mutant strains, offering a powerful platform for dissecting stress response signaling and identifying antifungal compounds that target the CWI pathway.

Cutinases are versatile enzymes with substrate promiscuity, making them promising candidates for the degradation of both natural and synthetic polyesters. While bacterial cutinases have been extensively studied, fungal cutinases remain underexplored, particularly in their enzymatic activity beyond their role in plant virulence. In this study, we investigated four cutinases from two Fusarium strains. Both strains displayed activity on Impranil-DLN, revealing their polyester-degrading potential. The strains were identified as Fusarium oxysporum (strain 38) and Fusarium redolens (strain 62). We characterized two cutinases per strain, tentatively called Cut1 and Cut5. Phylogenetic analysis revealed that both Cut1 clustered together in one branch where the Cut5 variants are closely related to the previously characterized bis(2-hydroxyethyl) terephthalate (BHET)-hydrolyzing enzyme FoCut5, providing structural insights and insights into their catalytic potential. We successfully expressed the Cut5 cutinases in Aspergillus niger using a CRISPR-Cas9-based multicopy integration system, resulting in enhanced degradation of Impranil-DLN and tributyrin. Using the same multicopy integration approach, transformants containing multicopy Cut1 variants were obtained but found to produce considerably lower amounts of Cut1, resulting in less activity and disabling further purification. The optimal substrate length, temperature, and pH for both Cut5 enzymes were determined. Additionally, we show activity of the purified Cut5 enzymes on synthetic substrates Impranil-DLN and BHET, suggesting that these fungal cutinases may be valuable for bioremediation. Accelerating the discovery of fungal cutinases and optimizing their expression systems holds promise for future strategies for polymer degradation to reduce agricultural and plastic waste. IMPORTANCE Cutinases are promising enzymes for a spectrum of applications due to their substrate promiscuity toward both natural and synthetic polymers. This makes them strong candidates for the development of sustainable solutions to battle environmental pollution. Therefore, the successful production and characterization of novel cutinases is fundamental for understanding their mechanisms and potential applications. In this study, we have identified, produced, and characterized two cutinases from different Fusarium species using a multicopy integration system in Aspergillus niger. Structural characteristics and in vivo and in vitro enzyme activity provide a unique insight into the polyester-degrading activity of these enzymes and how they can contribute to more sustainable solutions to our current waste management and pollution challenges.

Sorbicillinoids are yellow secondary metabolites synthesized through an elegant combination of enzymatic and spontaneous biochemical processes. The flavin-dependent monooxygenase SorC and oxidase SorD are crucial in this interplay, enabling the generation of a diverse array of functionally complex sorbicillinoids. By solving the crystal structures of SorC and SorD from Penicillium chrysogenum with sorbicillin bound in the active site, we describe the catalytically active binding conformations, crucial for attaining enantioselective and stereoselective control in these enzymatic reactions. The structure of SorC was resolved with the cofactor FAD in its out state, which allowed us to identify key residues that modulate flavin mobility and other conformational changes. Catalytic residues of SorC were also confirmed by detailed characterization of wild-type and several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome integration system, we could heterologously express the flavin-dependent oxidase SorD from P. chrysogenum in Aspergillus niger with high yields and purity. This allowed us to obtain the crystal structure of SorD with sorbicillin bound in a viable catalytic conformation. Structural analysis of the obtained complex provided insights into the substrate binding pose and highlighted potentially critical active site residues. Ultimately, having both SorC and SorD at our disposal enabled us to investigate their functions and interplays in the biosynthesis of a vast array of functionally complex sorbicillinoids.

Many microorganisms are able to use plant-derived aromatic and cyclic compounds like the common plant secondary metabolite quinic acid as carbon and energy sources. In fungi, 3 enzymatic steps convert quinic acid into the common intermediate protocatechuic acid, which is then further converted into tricarboxylic acid cycle intermediates. The genes encoding these 3 enzymes are known to be part of a gene cluster in Neurospora crassa along with a permease, a gene of unknown function, and an activator-repressor module controlling expression of the cluster. This gene cluster is conserved in fungi and has also been studied in Aspergillus nidulans, where an additional gene of unknown function is included. Here, we studied these genes in the filamentous fungus Aspergillus niger, where the availability of high-quality, well-annotated genomes and efficient tools for genome-editing and global gene expression analysis could provide new insights into quinic acid utilization in fungi. Using homology and whole transcriptome sequencing, we identified the genes involved in quinic acid utilization. Knockout mutants of these genes were then created to observe the growth phenotype on quinic acid media. We showed that not all the genes involved in quinic acid utilization in A. niger are linked. In addition to the in-cluster permease gene, we identified a second, previously unknown off-cluster permease gene which was upregulated in the presence of quinic acid. These 2 permeases were determined to function optimally at different pH levels, with the in-cluster permease being more effective at pH 6.5 and the off-cluster permease more effective at pH 3.5.

Multiple paralogs are found in the fungal genomes for several genes that encode proteins involved in cell wall biosynthesis. The genome of A. niger contains five genes encoding putative α-1,3-glucan synthases (AgsA-E) and seven genes encoding putative glucan-chitin crosslinking enzymes (CrhA-G). Here, we systematically studied the effects of the deletion of single (agsA or agsE), double (agsA and agsE), or all five ags genes (agsA-E) present in A. niger. Morphological and biochemical analysis of ags mutants emphasizes the important role of agsE in cell wall integrity, while deletion of other ags genes had minimal impact. Loss of agsE compromised cell wall integrity and altered pellet morphology in liquid cultures.Previous studies have indicated that deletion of all crh genes in A. niger did not result in cell wall integrity-related phenotypes. To determine whether the ags and crh gene families have redundant functions, both gene families were deleted using iterative CRISPR/Cas9 mediated genome editing. The 12-fold deletion mutant was viable and did not exhibit growth defects under non-stressing growth conditions. A synergistic effect on cell wall integrity was observed in this 12-fold deletion mutant, particularly when exposed to cell wall-perturbing compounds. The cell wall composition, extractability of glucans by alkali, and scanning electron microscopy analysis showed no differences between the parental strain and mutants lacking ags genes, crh genes, or both. These observations underscore the ability of fungal cells to adapt and secure cell wall integrity, even when two entire cell wall protein-encoding gene families are missing.

Abstract Background Aspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI. Results Here we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent. Conclusion The awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

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.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium. Bacterial-fungal interactions can stimulate the production of specialised microbial metabolites. Here, Richter et al. use co-culture experimental evolution to show that the presence of a fungus selects for increased surfactin production in the bacterium Bacillus subtilis, which inhibits fungal growth and facilitates the competitive success of the bacterium.

The glycoside hydrolase family 3 (GH3) β-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 β-glucosidases. Two distinct configurations were identified in fungal GH3 β-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in β-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger β-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric β-glucosidases enzymes for industrial application.

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. 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. 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.

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.

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 β-ketoadipate which is further cleaved to succinyl-CoA and acetyl-CoA. These β-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 β-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 β-ketoadipate:succinyl-CoA transferase; and NRRL3_01526 (kctA) encodes β-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 β-ketoadipate.

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 & mu;g & BULL;mL(-1) PatE protein in the culture medium with a purity just under 90%.

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.

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.

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.

Tannic acid, a hydrolysable gallotannin present in plant tissues, consists of a central glucose molecule esterified with gallic acid molecules. Some microorganisms, including severalAspergillusspecies, 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)2Cys6transcriptional 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 ΔtanXmutant. 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, inA. nigergallic acid utilization involves a hitherto unknown pathway. Transcriptome analysis of the ΔtanXmutant 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_04659mutant 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.

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.

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.