Publications by authors named "Mark Arentshorst"

54 Publications

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

Microorganisms 2020 Dec 2;8(12). Epub 2020 Dec 2.

Institute of Biology Leiden, Microbial Sciences, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

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 strain in which the gene was deleted. Previous work has shown that the deletion of causes hyper-branching and thicker cell walls, traits that may be beneficial for the reduction in fermentation viscosity and lysis. Hyper-branching of 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 in liquid medium. Morphological analysis showed that formed wild type-like pellets at pH 5.0, whereas the hyper-branching 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 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.
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http://dx.doi.org/10.3390/microorganisms8121918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761569PMC
December 2020

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

Cell Surf 2020 Dec 21;6:100039. Epub 2020 Mar 21.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands.

Chitin is an important fungal cell wall component that is cross-linked to β-glucan for structural integrity. Acquisition of chitin to glucan cross-links has previously been shown to be performed by transglycosylation enzymes in , called Congo Red hypersensitive (Crh) enzymes. Here, we characterized the impact of deleting all seven members of the gene family () in on cell wall integrity, cell wall composition and genome-wide gene expression. In this study, we show that the seven-fold 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 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 gene family in 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.
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http://dx.doi.org/10.1016/j.tcsw.2020.100039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389268PMC
December 2020

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

Gene X 2020 Dec 28;5:100028. Epub 2020 Jan 28.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands.

Post-fermentation fungal biomass waste provides a viable source for chitin. Cell wall chitin of filamentous fungi, and in particular its de-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 . 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 (), encodes an orthologue of Bypass of (), 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.
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http://dx.doi.org/10.1016/j.gene.2020.100028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285910PMC
December 2020

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

Fungal Genet Biol 2020 Jun 3;139:103377. Epub 2020 Apr 3.

Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands. Electronic address:

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)Cys 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.
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http://dx.doi.org/10.1016/j.fgb.2020.103377DOI Listing
June 2020

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

Fungal Genet Biol 2020 03 27;136:103319. Epub 2019 Dec 27.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, the Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.fgb.2019.103319DOI Listing
March 2020

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

Fungal Biol Biotechnol 2019 21;6:13. Epub 2019 Sep 21.

1Department Molecular Microbiology and Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

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 -genes encoding putative glucan-chitin crosslinking enzymes involved in cell wall biosynthesis.

Results: Here, we present a CRISPR/Cas9 system for 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 -gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker () 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 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.
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http://dx.doi.org/10.1186/s40694-019-0076-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754632PMC
September 2019

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

Arch Microbiol 2020 Jan 1;202(1):197-203. Epub 2019 Aug 1.

Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

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.
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http://dx.doi.org/10.1007/s00203-019-01709-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949202PMC
January 2020

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

ACS Cent Sci 2019 Jun 24;5(6):1067-1078. Epub 2019 May 24.

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

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 β-xylosidases and -β-1,4-xylanases in the secretomes of , by the use of chemical probes inspired by the β-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.
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http://dx.doi.org/10.1021/acscentsci.9b00221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598175PMC
June 2019

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

Appl Microbiol Biotechnol 2019 06;103(12):5063

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333, BE, Leiden, The Netherlands.

The correct title is: Mutations in AraR leading to constitutive expression of arabinolytic genes in Aspergillus niger under derepressing conditions.
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http://dx.doi.org/10.1007/s00253-019-09853-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828049PMC
June 2019

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

Appl Microbiol Biotechnol 2019 05 8;103(10):4125-4136. Epub 2019 Apr 8.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

The AraR transcription factor of Aspergillus niger encodes a Zn(II)Cys 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 AraR 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 AraR mutant was found to be sensitive to carbon catabolite repression, indicating that the CreA-mediated carbon catabolite repression is dominant over the AraR mutant allele. These mutations in AraR provide new opportunities to improve arabinase production in industrial fungal strains.
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http://dx.doi.org/10.1007/s00253-019-09777-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486530PMC
May 2019

FlbA-Regulated Gene Is Involved in Stress Resistance and Impacts Protein Secretion when Is Grown on Xylose.

Appl Environ Microbiol 2019 01 9;85(2). Epub 2019 Jan 9.

Microbiology, Department of Biology, Utrecht University, Utrecht, The Netherlands

Proteins are secreted throughout the mycelium of except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene results in mycelial colonies that secrete proteins throughout the colony. However, Δ strain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which, , was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the Δ deletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when was grown on xylose. Moreover, the Δ strain showed decreased resistance to HO and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when is grown on xylose. secretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production in .
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http://dx.doi.org/10.1128/AEM.02282-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328776PMC
January 2019

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

Microbiologyopen 2019 05 8;8(5):e00732. Epub 2018 Oct 8.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands.

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/GaaR revealed important differences in nuclear accumulation of N- versus C-terminally eGFP-tagged GaaR.
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http://dx.doi.org/10.1002/mbo3.732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528562PMC
May 2019

Parasexual Crossings for Bulk Segregant Analysis in Aspergillus niger to Facilitate Mutant Identification Via Whole Genome Sequencing.

Methods Mol Biol 2018 ;1775:277-287

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands.

The industrially important fungus Aspergillus niger is known to reproduce only asexually. The parasexual cycle of fungi can be used for crossing two different strains to produce segregants or progeny with combined mutations even in fungi without a known sexual cycle. In A. niger, the parasexual cycle has been extensively used to establish linkage groups and to generate genetic maps. With the advent of whole genome sequencing, the parasexual cycle has received renewed attention as a method to create segregants for bulk segregant analysis. Bulk segregant analysis is a genetic technique used to link and ultimately identify the mutation associated with a particular phenotype. In this chapter we describe the procedure for setting up parasexual crossings in A. niger. The segregants obtained with this method can be used in combination with next-generation sequencing to map mutations in the organism.
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http://dx.doi.org/10.1007/978-1-4939-7804-5_22DOI Listing
February 2019

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

Appl Microbiol Biotechnol 2018 Mar 24;102(6):2723-2736. Epub 2018 Jan 24.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

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.
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http://dx.doi.org/10.1007/s00253-018-8753-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847190PMC
March 2018

The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production.

Antonie Van Leeuwenhoek 2018 Mar 30;111(3):311-322. Epub 2017 Sep 30.

Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Aspergillus niger secretes proteins throughout the colony except for the zone that forms asexual spores called conidia. Inactivation of flbA that encodes a regulator of G-protein signaling results in colonies that are unable to reproduce asexually and that secrete proteins throughout the mycelium. In addition, the ΔflbA strain shows cell lysis and has thinner cell walls. Expression analysis showed that 38 predicted transcription factor genes are differentially expressed in strain ΔflbA. Here, the most down-regulated predicted transcription factor gene, called fum21, was inactivated. Growth, conidiation, and protein secretion were not affected in strain Δfum21. Whole genome expression analysis revealed that 63 and 11 genes were down- and up-regulated in Δfum21, respectively, when compared to the wild-type strain. Notably, 24 genes predicted to be involved in secondary metabolism were down-regulated in Δfum21, including 10 out of 12 genes of the fumonisin cluster. This was accompanied by absence of fumonisin production in the deletion strain and a 25% reduction in production of pyranonigrin A. Together, these results link FlbA-mediated sporulation-inhibited secretion with mycotoxin production.
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http://dx.doi.org/10.1007/s10482-017-0952-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816093PMC
March 2018

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

FEBS Lett 2017 05 6;591(10):1408-1418. Epub 2017 May 6.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, The Netherlands.

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.
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http://dx.doi.org/10.1002/1873-3468.12654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488244PMC
May 2017

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

Genetics 2017 01 9;205(1):169-183. Epub 2016 Nov 9.

Molecular Microbiology and Biotechnology, Leiden University, Institute of Biology Leiden, 2333 BE Leiden, The Netherlands

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.
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http://dx.doi.org/10.1534/genetics.116.194050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223501PMC
January 2017

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

Microb Cell Fact 2016 Aug 20;15(1):145. Epub 2016 Aug 20.

Department Applied and Molecular Microbiology, Institute of Biotechnology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.

Background: The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency.

Results: In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein.

Conclusion: Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.
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http://dx.doi.org/10.1186/s12934-016-0543-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992228PMC
August 2016

Aspergillus fumigatus MADS-Box Transcription Factor rlmA Is Required for Regulation of the Cell Wall Integrity and Virulence.

G3 (Bethesda) 2016 09 8;6(9):2983-3002. Epub 2016 Sep 8.

Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, 13565 São Carlos, São Paulo, Brazil

The Cell Wall Integrity (CWI) pathway is the primary signaling cascade that controls the de novo synthesis of the fungal cell wall, and in Saccharomyces cerevisiae this event is highly dependent on the RLM1 transcription factor. Here, we investigated the function of RlmA in the fungal pathogen Aspergillus fumigatus We show that the ΔrlmA strain exhibits an altered cell wall organization in addition to defects related to vegetative growth and tolerance to cell wall-perturbing agents. A genetic analysis indicated that rlmA is positioned downstream of the pkcA and mpkA genes in the CWI pathway. As a consequence, rlmA loss-of-function leads to the altered expression of genes encoding cell wall-related proteins. RlmA positively regulates the phosphorylation of MpkA and is induced at both protein and transcriptional levels during cell wall stress. The rlmA was also involved in tolerance to oxidative damage and transcriptional regulation of genes related to oxidative stress adaptation. Moreover, the ΔrlmA strain had attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Our results suggest that RlmA functions as a transcription factor in the A. fumigatus CWI pathway, acting downstream of PkcA-MpkA signaling and contributing to the virulence of this fungus.
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http://dx.doi.org/10.1534/g3.116.031112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015955PMC
September 2016

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

Cell Microbiol 2016 09 29;18(9):1268-84. Epub 2016 Jul 29.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

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 β-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.
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http://dx.doi.org/10.1111/cmi.12624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129474PMC
September 2016

A set of isogenic auxotrophic strains for constructing multiple gene deletion mutants and parasexual crossings in Aspergillus niger.

Arch Microbiol 2016 Nov 1;198(9):861-8. Epub 2016 Jun 1.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

To construct a set of isogenic auxotrophic strains in Aspergillus niger suited for creating multiple gene deletion mutants and executing parasexual crossings, we have combined mutations in genes involved in colour pigmentation (fwnA and olvA) with well-selectable auxotrophic markers (pyrG, nicB, argB, and adeA). All markers, except for the pyrG marker, were introduced by targeted deletion, omitting UV mutagenesis of the strains. Aspergillus oryzae orthologous genes of the argB, nicB, and adeA markers were used as heterologous selection markers, and all markers were shown to complement to respective auxotrophic A. niger mutants. A quadruple auxotrophic marker was further constructed suitable for multiple gene deletions. Genome sequencing of two auxotrophic colour mutants JN3.2 (olvA::pyrG, argB::hygB) and JN6.2 (olvA::pyrG, nicB::hygB) revealed four SNPs between them in non-coding regions, indicating a high level of isogenicity between both strains. The availability of near-isogenic complementary auxotrophic colour mutants facilitates the selection of diploids and the isolation of haploid segregants from the diploid using the parasexual cycle.
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http://dx.doi.org/10.1007/s00203-016-1240-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040738PMC
November 2016

Improving cellulase production by Aspergillus niger using adaptive evolution.

Biotechnol Lett 2016 Jun 15;38(6):969-74. Epub 2016 Feb 15.

Fungal Molecular Physiology, CBS-KNAW Fungal Biodiversity Centre, Utrecht University, Utrecht, The Netherlands.

Objectives: To evaluate the potential of adaptive evolution as a tool in generating strains with an improved production of plant biomass degrading enzymes.

Results: An Aspergillus niger cellulase mutant was obtained by adaptive evolution. Physiological properties of this mutant revealed a five times higher cellulose production than the parental strain. Transcriptomic analysis revealed that the expression of noxR, encoding the regulatory subunit of the NADPH oxidase complex, was reduced in the mutant compared to the parental strain. Subsequent analysis of a noxR knockout strain showed the same phenotypic effect as observed for the evolution mutant, confirming the role of NoxR in cellulose degradation.

Conclusions: Adaptive evolution is an efficient approach to modify a strain and activate genes involved in polysaccharide degradation.
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http://dx.doi.org/10.1007/s10529-016-2060-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853455PMC
June 2016

I-SceI enzyme mediated integration (SEMI) for fast and efficient gene targeting in Trichoderma reesei.

J Biotechnol 2016 Mar 6;222:25-8. Epub 2016 Feb 6.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands. Electronic address:

We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequencies by combining I-SceI mediated double strand break with disruption of the tku70 gene. The inability of the tku70 mutant to repair a I-SceI mediated DSB via NHEJ was used to force integration of an expression cassette with homologous flanks surrounding the DSB site. Besides expressing I-SceI from a plasmid, we also show that adding I-SceI enzyme during transformation was successful to generate DSBs. The I-SceI enzyme mediated integration, or SEMI, in combination with a Δtku70 mutant has a synergistic effect on homologous recombination efficiencies as 90-100% of the transformants exhibited integration of the expression cassette at the homologous site.
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http://dx.doi.org/10.1016/j.jbiotec.2016.02.012DOI Listing
March 2016

Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites.

G3 (Bethesda) 2015 Nov 13;6(1):193-204. Epub 2015 Nov 13.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, 2333 BE, Leiden, The Netherlands

The asexual filamentous fungus Aspergillus niger is an important industrial cell factory for citric acid production. In this study, we genetically characterized a UV-generated A. niger mutant that was originally isolated as a nonacidifying mutant, which is a desirable trait for industrial enzyme production. Physiological analysis showed that this mutant did not secrete large amounts of citric acid and oxalic acid, thus explaining the nonacidifying phenotype. As traditional complementation approaches to characterize the mutant genotype were unsuccessful, we used bulk segregant analysis in combination with high-throughput genome sequencing to identify the mutation responsible for the nonacidifying phenotype. Since A. niger has no sexual cycle, parasexual genetics was used to generate haploid segregants derived from diploids by loss of whole chromosomes. We found that the nonacidifying phenotype was caused by a point mutation in the laeA gene. LaeA encodes a putative methyltransferase-domain protein, which we show here to be required for citric acid production in an A. niger lab strain (N402) and in other citric acid production strains. The unexpected link between LaeA and citric acid production could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also involved in regulating the production of secondary metabolites. Finally, we show that our systems genetics approach is a powerful tool to identify trait mutations.
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http://dx.doi.org/10.1534/g3.115.024067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704718PMC
November 2015

I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

Appl Microbiol Biotechnol 2015 Dec 15;99(23):10083-95. Epub 2015 Aug 15.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Kluyver Centre for Genomics of Industrial Fermentation, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.
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http://dx.doi.org/10.1007/s00253-015-6829-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643118PMC
December 2015

The interaction of induction and repression mechanisms in the regulation of galacturonic acid-induced genes in Aspergillus niger.

Fungal Genet Biol 2015 Sep 27;82:32-42. Epub 2015 Jun 27.

Leiden University, Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands. Electronic address:

Aspergillus niger is an important industrial fungus expressing a broad spectrum of pectinolytic genes. The main constituent of pectin, polygalacturonic acid (PGA), is degraded into galacturonic acid (GA) by the combined activity of endo- and exo-polygalacturonases some of which are specifically induced by GA. The regulatory mechanisms that control the expression of genes encoding PGA-degrading enzymes are not well understood. Based on available genome-wide expression profiles from literature, we selected five genes that were specifically induced by GA. These genes include three exo-polygalacturonases (pgaX, pgxB and pgxC), a GA transporter (gatA), and an intracellular enzyme involved in GA metabolism (gaaB). These five genes contain a conserved motif (5'-TCCNCCAAT-3') in their promoter regions, which we named GARE (galacturonic acid-responsive element). Promoter deletion studies and site-directed mutagenesis of the conserved motif of the pgaX gene showed that the conserved element is required for GA-mediated induction. A set of promoter reporter strains was constructed by fusing the promoter region of the five above-mentioned genes to the amdS reporter gene. Expression of the amdS gene is quantitatively correlated with ability to utilise acetamide as an N-source, hence higher expression of amdS improves growth of the strain on acetamide and therefore can be used as an in vivo reporter for gene expression. Growth analysis of the reporter strains indicated that four genes (pgaX, pgxB, pgxC, and gatA) are specifically induced by GA. The in vivo promoter reporter strains were also used to monitor carbon catabolite repression control. Except for gaaB, all promoter-reporter genes analysed were repressed by glucose in a glucose concentration-dependent way. Interestingly, the strength of glucose repression was different for the tested promoters. CreA is important in mediating carbon catabolite repression as deletion of the creA gene in the reporter strains abolished carbon catabolite repression for most promoters. Interestingly, the pgxC promoter was still repressed by glucose even in the creA null background, suggesting a role for alternative repression mechanisms. Finally, we showed that low concentrations of GA are required to induce gene expression of pgaX, pgxB, and pgxC even under derepressing conditions. The results obtained are consistent with a model in which a GA-specific transcription factor is activated by GA or a GA-derivative, which binds to the conserved motif, possibly in combination with the HAP-complex, to drive GA-specific gene expression.
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http://dx.doi.org/10.1016/j.fgb.2015.06.006DOI Listing
September 2015

A new vector for efficient gene targeting to the locus in .

Fungal Biol Biotechnol 2015 14;2. Epub 2015 Mar 14.

Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

Background: The possibility for efficient gene targeting for the controlled integration of DNA constructs is an important tool in fungal genetics.

Findings: In this study, we report a new targeting vector based on the marker in . The DNA sequence to be targeted is surrounded by two fragments of the gene to allow homologous recombination of the recombinant DNA at the locus. The 5' end of the targeting cassette contains a non-functional truncated open reading frame (first 112 bases deleted) and the 3' untranslated region (3' UTR). At the 3' end, the targeting cassette consists of the 3' flanking region of the gene. A unique I site between the flanks allows the insertion of a gene of interest. The linearized targeting cassette is transformed to the mutant strain AB4.1 or a derivative thereof. By using a constitutively expressed luciferase reporter gene () as an example, it is shown that the targeting system is efficient as 4 out of 6 (67%) AB4.1 transformants and 51 out of 66 (77%) MA169.4 ( ) transformants contained the reporter gene at the locus. A luciferase (lux) activity assay, performed with independently obtained transformants in which the reporter was integrated at the locus, showed comparable and reproducible lux activities.

Conclusion: The new targeting vector is an important improvement to the existing method for gene targeting in Although the vector is specific for the presented design and approach is easily applicable for constructing integration vectors for other fungi.
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http://dx.doi.org/10.1186/s40694-015-0012-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611571PMC
March 2015

Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in .

Fungal Biol Biotechnol 2014 14;1. Epub 2014 Oct 14.

Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden University, Sylviusweg 72, Leiden, 2333 BE The Netherlands.

Background: Galactofuranose (Gal)-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Gal-containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in is characterized by the specific induction of the gene, which encodes a cell wall α-glucan synthase.

Results: In this study, we screened a collection of cell wall mutants with an induced expression of for defects in Gal biosynthesis using a with anti-Gal antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene (). Here, we have identified six additional UDP-galactopyranose mutase () mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Gal-levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of resulted in an even more severe reduction of Gal in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The gene is also required for growth on galactose despite the presence of two UgeA homologs in the genome.

Conclusion: By using a classical mutant screen and whole genome sequencing of a new Gal-deficient mutant, the UDP-glucose-4-epimerase gene () has been identified. UgeA is required for the biosynthesis of Gal as well as for galactose metabolism in .
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http://dx.doi.org/10.1186/s40694-014-0006-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5598270PMC
October 2014

Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi.

Microbiology (Reading) 2014 Feb 2;160(Pt 2):316-329. Epub 2013 Dec 2.

Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The Netherlands.

The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in Saccharomyces cerevisiae, was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p orthologue in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in S. cerevisiae is lethal. Similarly, deletion of the A. niger orthologue of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (encoding SecA, SecH and SsoA the A. niger orthologues of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similar to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.
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http://dx.doi.org/10.1099/mic.0.074252-0DOI Listing
February 2014

The transcriptional repressor TupA in Aspergillus niger is involved in controlling gene expression related to cell wall biosynthesis, development, and nitrogen source availability.

PLoS One 2013 29;8(10):e78102. Epub 2013 Oct 29.

Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Leiden, The Netherlands.

The Tup1-Cyc8 (Ssn6) complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA) homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis with a genomic library showed that the tupA gene could complement the phenotypes of the mutant. Screening of a collection of 240 mutants with constitutive expression of agsA identified sixteen additional pigment-secreting mutants, which were all mutated in the tupA gene. The phenotypes of the tupA mutants were very similar to the phenotypes of a tupA deletion strain. Further analysis of the tupA-17 mutant and the ΔtupA mutant revealed that TupA is also required for normal growth and morphogenesis. The production of the pigment at 37°C is nitrogen source-dependent and repressed by ammonium. Genome-wide expression analysis of the tupA mutant during exponential growth revealed derepression of a large group of diverse genes, including genes related to development and cell wall biosynthesis, and also protease-encoding genes that are normally repressed by ammonium. Comparison of the transcriptome of up-regulated genes in the tupA mutant showed limited overlap with the transcriptome of caspofungin-induced cell wall stress-related genes, suggesting that TupA is not a general suppressor of cell wall stress-induced genes. We propose that TupA is an important repressor of genes related to development and nitrogen metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078102PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812127PMC
September 2014