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.

Intraspecific variability in heat resistance of fungal conidia.

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.

High sorbic acid resistance of Penicillium roqueforti is mediated by the SORBUS gene cluster.

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 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.

Genome sequences of 24 Aspergillus niger sensu stricto strains to study strain diversity, heterokaryon compatibility, and sexual reproduction.

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 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.

Toward Microbial Recycling and Upcycling of Plastics: Prospects and Challenges.

Annually, 400 Mt of plastics are produced of which roughly 40% is discarded within a year. Current plastic waste management approaches focus on applying physical, thermal, and chemical treatments of plastic polymers. However, these methods have severe limitations leading to the loss of valuable materials and resources. Another major drawback is the rapid accumulation of plastics into the environment causing one of the biggest environmental threats of the twenty-first century. Therefore, to complement current plastic management approaches novel routes toward plastic degradation and upcycling need to be developed. Enzymatic degradation and conversion of plastics present a promising approach toward sustainable recycling of plastics and plastics building blocks. However, the quest for novel enzymes that efficiently operate in cost-effective, large-scale plastics degradation poses many challenges. To date, a wide range of experimental set-ups has been reported, in many cases lacking a detailed investigation of microbial species exhibiting plastics degrading properties as well as of their corresponding plastics degrading enzymes. The apparent lack of consistent approaches compromises the necessary discovery of a wide range of novel enzymes. In this review, we discuss prospects and possibilities for efficient enzymatic degradation, recycling, and upcycling of plastics, in correlation with their wide diversity and broad utilization. Current methods for the identification and optimization of plastics degrading enzymes are compared and discussed. We present a framework for a standardized workflow, allowing transparent discovery and optimization of novel enzymes for efficient and sustainable plastics degradation in the future.

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.

Genome sequencing of the neotype strain CBS 554.65 reveals the MAT1-2 locus of Aspergillus niger.

BACKGROUND: Aspergillus 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. RESULTS: The 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. CONCLUSIONS: This 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.

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.

Preservation stress resistance of melanin deficient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing.

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.

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.

Correction: Glycosylated cyclophellitol-derived activity-based probes and inhibitors for cellulases.

[This corrects the article DOI: 10.1039/D0CB00045K.].

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.

Carbohydrate Binding Modules: Diversity of Domain Architecture in Amylases and Cellulases From Filamentous Microorganisms.

Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.

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.