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.

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.

Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions [corrected].

The AraR transcription factor of Aspergillus niger encodes a Zn(II)2Cys6 transcription factor required for the induction of genes encoding arabinolytic enzymes. One of the target genes of AraR is abfA, encoding an arabinofuranosidase. The expression of abfA as well as other L-arabinose-induced genes in A. niger requires the presence of L-arabinose or its derivative L-arabitol as an inducer to activate AraR-dependant gene expression. In this study, mutants were isolated that express L-arabinose-induced genes independently of the presence of an inducer under derepressing conditions. To obtain these mutants, a reporter strain was constructed in a ΔcreA background containing the L-arabinose-responsive promoter (PabfA) fused to the acetamidase (amdS) gene. Spores of the ΔcreA PabfA-amdS reporter strain were UV-mutagenized and mutants were obtained by their ability to grow on acetamide without the presence of inducer. From a total of 164 mutants, 15 mutants were identified to contain transacting mutations resulting in high arabinofuranosidase activity in the medium after growth under non-inducing conditions. Sequencing of the araR gene of the 15 constitutive mutants revealed that 14 mutants carried a mutation in AraR. Some mutations were found more than once and in total nine different point mutations were identified in AraR. The AraRN806I point mutation was reintroduced into a parental strain and confirmed that this point mutation leads to inducer-independent expression of AraR target genes. The inducer independent of L-arabinose-induced genes in the AraRN806I mutant was found to be sensitive to carbon catabolite repression, indicating that the CreA-mediated carbon catabolite repression is dominant over the AraRN806I mutant allele. These mutations in AraR provide new opportunities to improve arabinase production in industrial fungal strains.

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.

Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid-responsive transcription factor gaaR.

The transcription factor GaaR is needed for the expression of genes required for pectin degradation and transport and catabolism of the main degradation product, D-galacturonic acid (GA) in Aspergillus niger. In this study, we used the strong constitutive gpdA promoter of Aspergillus nidulans to overexpress gaaR in A. niger. Overexpression of gaaR resulted in an increased transcription of the genes encoding pectinases, (putative) GA transporters, and catabolic pathway enzymes even under non-inducing conditions, i.e., in the absence of GA. Exoproteome analysis of a strain overexpressing gaaR showed that this strain secretes highly elevated levels of pectinases when grown in fructose. The genes encoding exo-polygalacturonases were found to be subjected to CreA-mediated carbon catabolite repression, even in the presence of fructose. Deletion of creA in the strain overexpressing gaaR resulted in a further increase in pectinase production in fructose. We showed that GaaR localizes mainly in the nucleus regardless of the presence of an inducer, and that overexpression of gaaR leads to an increased concentration of GaaR in the nucleus.

Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis.

The biosynthesis of cell surface-located galactofuranose (Galf)-containing glycostructures such as galactomannan, N-glycans and O-glycans in filamentous fungi is important to secure the integrity of the cell wall. UgmA encodes an UDP-galactopyranose mutase, which is essential for the formation of Galf. Consequently, the ΔugmA mutant lacks Galf-containing molecules. Our previous work in Aspergillus niger work suggested that loss of function of ugmA results in activation of the cell wall integrity (CWI) pathway which is characterized by increased expression of the agsA gene, encoding an α-glucan synthase. In this study, the transcriptional response of the ΔugmA mutant was further linked to the CWI pathway by showing the induced and constitutive phosphorylation of the CWI-MAP kinase in the ΔugmA mutant. To identify genes involved in cell wall remodelling in response to the absence of galactofuranose biosynthesis, a genome-wide expression analysis was performed using RNAseq. Over 400 genes were higher expressed in the ΔugmA mutant compared to the wild-type. These include genes that encode enzymes involved in chitin (gfaB, gnsA, chsA) and α-glucan synthesis (agsA), and in ß-glucan remodelling (bgxA, gelF and dfgC), and also include several glycosylphosphatidylinositol (GPI)-anchored cell wall protein-encoding genes. In silico analysis of the 1-kb promoter regions of the up-regulated genes in the ΔugmA mutant indicated overrepresentation of genes with RlmA, MsnA, PacC and SteA-binding sites. The importance of these transcription factors for survival of the ΔugmA mutant was analysed by constructing the respective double mutants. The ΔugmA/ΔrlmA and ΔugmA/ΔmsnA double mutants showed strong synthetic growth defects, indicating the importance of these transcription factors to maintain cell wall integrity in the absence of Galf biosynthesis.

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.

W361R mutation in GaaR, the regulator of D-galacturonic acid-responsive genes, leads to constitutive production of pectinases in Aspergillus niger.

Polysaccharides present in plant biomass, such as pectin, are the main carbon source for filamentous fungi. Aspergillus niger naturally secretes pectinases to degrade pectin and utilize the released monomers, mainly D-galacturonic acid. The transcriptional activator GaaR, the repressor of D-galacturonic acid utilization GaaX, and the physiological inducer 2-keto-3-deoxy-L-galactonate play important roles in the transcriptional regulation of D-galacturonic acid-responsive genes, which include the genes encoding pectinases. In this study, we described the mutations found in gaaX and gaaR that enabled constitutive (i.e., inducer-independent) expression of pectinases by A. niger. Using promoter-reporter strains (PpgaX-amdS) and polygalacturonic acid plate assays, we showed that W361R mutation in GaaR results in constitutive production of pectinases. Analysis of subcellular localization of C-terminally eGFP-tagged GaaR/GaaRW361R revealed important differences in nuclear accumulation of N- versus C-terminally eGFP-tagged GaaR.

Modulating Transcriptional Regulation of Plant Biomass Degrading Enzyme Networks for Rational Design of Industrial Fungal Strains.

Filamentous fungi are the most important microorganisms for the industrial production of plant polysaccharide degrading enzymes due to their unique ability to secrete these proteins efficiently. These carbohydrate active enzymes (CAZymes) are utilized industrially for the hydrolysis of plant biomass for the subsequent production of biofuels and high-value biochemicals. The expression of the genes encoding plant biomass degrading enzymes is tightly controlled. Naturally, large amounts of CAZymes are produced and secreted only in the presence of the plant polysaccharide they specifically act on. The signal to produce is conveyed via so-called inducer molecules which are di- or mono-saccharides (or derivatives thereof) released from the specific plant polysaccharides. The presence of the inducer results in the activation of a substrate-specific transcription factor (TF), which is required not only for the controlled expression of the genes encoding the CAZymes, but often also for the regulation of the expression of the genes encoding sugar transporters and catabolic pathway enzymes needed to utilize the released monosaccharide. Over the years, several substrate-specific TFs involved in the degradation of cellulose, hemicellulose, pectin, starch and inulin have been identified in several fungal species and systems biology approaches have made it possible to uncover the enzyme networks controlled by these TFs. The requirement for specific inducers for TF activation and subsequently the expression of particular enzyme networks determines the choice of feedstock to produce enzyme cocktails for industrial use. It also results in batch-to-batch variation in the composition and amounts of enzymes due to variations in sugar composition and polysaccharide decorations of the feedstock which hampers the use of cheap feedstocks for constant quality of enzyme cocktails. It is therefore of industrial interest to produce specific enzyme cocktails constitutively and independently of inducers. In this review, we focus on the methods to modulate TF activities for inducer-independent production of CAZymes and highlight various approaches that are used to construct strains displaying constitutive expression of plant biomass degrading enzyme networks. These approaches and combinations thereof are also used to construct strains displaying increased expression of CAZymes under inducing conditions, and make it possible to design strains in which different enzyme mixtures are simultaneously produced independently of the carbon source.

An Evolutionarily Conserved Transcriptional Activator-Repressor Module Controls Expression of Genes for D-Galacturonic Acid Utilization in Aspergillus niger.

The expression of genes encoding extracellular polymer-degrading enzymes and the metabolic pathways required for carbon utilization in fungi are tightly controlled. The control is mediated by transcription factors that are activated by the presence of specific inducers, which are often monomers or monomeric derivatives of the polymers. A D-galacturonic acid-specific transcription factor named GaaR was recently identified and shown to be an activator for the expression of genes involved in galacturonic acid utilization in Botrytis cinerea and Aspergillus niger Using a forward genetic screen, we isolated A. niger mutants that constitutively express GaaR-controlled genes. Reasoning that mutations in the gaaR gene would lead to a constitutively activated transcription factor, the gaaR gene in 11 of the constitutive mutants was sequenced, but no mutations in gaaR were found. Full genome sequencing of five constitutive mutants revealed allelic mutations in one particular gene encoding a previously uncharacterized protein (NRRL3_08194). The protein encoded by NRRL3_08194 shows homology to the repressor of the quinate utilization pathway identified previously in Neurospora crassa (qa-1S) and Aspergillus nidulans (QutR). Deletion of NRRL3_08194 in combination with RNA-seq analysis showed that the NRRL3_08194 deletion mutant constitutively expresses genes involved in galacturonic acid utilization. Interestingly, NRRL3_08194 is located next to gaaR (NRRL3_08195) in the genome. The homology to the quinate repressor, the chromosomal clustering, and the constitutive phenotype of the isolated mutants suggest that NRRL3_08194 is likely to encode a repressor, which we name GaaX. The GaaR-GaaX module and its chromosomal organization is conserved among ascomycetes filamentous fungi, resembling the quinate utilization activator-repressor module in amino acid sequence and chromosomal organization.

The pathway intermediate 2-keto-3-deoxy-L-galactonate mediates the induction of genes involved in D-galacturonic acid utilization in Aspergillus niger.

In Aspergillus niger, the enzymes encoded by gaaA, gaaB, and gaaC catabolize d-galacturonic acid (GA) consecutively into l-galactonate, 2-keto-3-deoxy-l-galactonate, pyruvate, and l-glyceraldehyde, while GaaD converts l-glyceraldehyde to glycerol. Deletion of gaaB or gaaC results in severely impaired growth on GA and accumulation of l-galactonate and 2-keto-3-deoxy-l-galactonate, respectively. Expression levels of GA-responsive genes are specifically elevated in the ∆gaaC mutant on GA as compared to the reference strain and other GA catabolic pathway deletion mutants. This indicates that 2-keto-3-deoxy-l-galactonate is the inducer of genes required for GA utilization.

The transcriptional activator GaaR of Aspergillus niger is required for release and utilization of d-galacturonic acid from pectin.

We identified the d-galacturonic acid (GA)-responsive transcriptional activator GaaR of the saprotrophic fungus, Aspergillus niger, which was found to be essential for growth on GA and polygalacturonic acid (PGA). Growth of the ΔgaaR strain was reduced on complex pectins. Genome-wide expression analysis showed that GaaR is required for the expression of genes necessary to release GA from PGA and more complex pectins, to transport GA into the cell, and to induce the GA catabolic pathway. Residual growth of ΔgaaR on complex pectins is likely due to the expression of pectinases acting on rhamnogalacturonan and subsequent metabolism of the monosaccharides other than GA.