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The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.

Genetic recombination is an important tool in strain breeding in many organisms. We studied the possibilities of mitotic recombination in strain breeding of the asexual fungus Aspergillus niger. By identifying genes that complemented mapped auxotrophic mutations, the physical map was compared to the genetic map of chromosome III using the genome sequence. In a program to construct a chromosome III-specific marker strain by selecting mitotic crossing-over in diploids, a mitotic recombination hotspot was identified. Analysis of the mitotic recombination hotspot revealed some physical features, elevated basal transcription and a possible correlation with purine stretches.

Contains fulltext : 51722.pdf (Publisher’s version ) (Closed access) The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

Background Filamentous fungi such as Aspergillus niger have a high capacity secretory system and are therefore widely exploited for the industrial production of native and heterologous proteins. However, in most cases the yields of non-fungal proteins are significantly lower than those obtained for fungal proteins. One well-studied bottleneck appears to be the result of mis-folding of heterologous proteins in the ER during early stages of secretion, with related stress responses in the host, including the unfolded protein response (UPR). This study aims at uncovering transcriptional and translational responses occurring in A. niger exposed to secretion stress. Results A genome-wide transcriptional analysis of protein secretion-related stress responses was determined using Affymetrix DNA GeneChips and independent verification for selected genes. Endoplasmic reticulum (ER)-associated stress was induced either by chemical treatment of the wild-type cells with dithiothreitol (DTT) or tunicamycin, or by expressing a human protein, tissue plasminogen activator (t-PA). All of these treatments triggered the UPR, as shown by the expression levels of several well-known UPR target genes. The predicted proteins encoded by most of the up-regulated genes function as part of the secretory system including chaperones, foldases, glycosylation enzymes, vesicle transport proteins, and ER-associated degradation proteins. Several genes were down-regulated under stress conditions and these included several genes that encode secreted enzymes. Moreover, translational regulation under ER stress was investigated by polysomal fractionation. This analysis confirmed the post-transcriptional control of hacA expression and highlighted that differential translation also occurs during ER stress, in particular for some genes encoding secreted proteins or proteins involved in ribosomal biogenesis and assembly. Conclusion This is first genome-wide analysis of both transcriptional and translational events following protein secretion stress. Insight has been gained into the molecular basis of protein secretion and secretion-related stress in an effective protein-secreting fungus, and provides an opportunity to identify target genes for manipulation in strain improvement strategies.

The two most abundant structural polysaccharides in nature are cellulose and the hemicellulose xylan, which are closely associated in plant cell walls (4). Filamentous fungi, particularly Aspergillus and Trichoderma species, are well-known and efficient producers of both cellulolytic and hemicellulolytic enzymes. The cellulase degradation system of these organisms consists of three classes of enzymes (2): endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). Members of all of these classes are necessary to degrade cellulose, a homopolymer of β-1,4-linked d-glucose. Xylan, however, is a heterogeneous polymer with a backbone consisting of β-1,4-linked d-xylose residues, which can be substituted at the C-2 and C-3 positions with various residues, such as acetic acid, α-l-arabinofuranose, (4-o-methyl)glucuronic acid, ferulic acid, and p-coumaric acid (5). Due to this heterogeneous composition, a more complex set of enzymes is required for xylan degradation. The following enzymes have been found to be necessary during the cooperative process of xylan breakdown: endoxylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), acetylxylan esterase (EC 3.1.1.72), α-l-arabinofuranosidase (EC 3.2.1.55), arabinoxylan arabinofuranohydrolase, β-glucuronidase (EC 3.2.1.139), feruloyl esterase, and p-coumaroyl esterase (3). The expression of cellulose- and xylan-degrading enzymes by Aspergillus and Trichoderma species has been studied extensively at the cellular level (1, 20, 21, 25). It has been shown that xylanase- and cellulase-encoding genes are regulated at the transcriptional level (10, 23, 31, 43). In the presence of d-glucose the genes are not expressed, and it has been shown that the carbon catabolite repressor protein CreA is involved in transcriptional repression of xylanase-encoding (10) and arabinase-encoding (38) genes in Aspergillus species. It has been demonstrated that in Trichoderma reesei the CreA counterpart Cre1 causes repression of transcription of cellulase-encoding (22, 23) and xylanase-encoding (30, 31) genes. However, far less is known about the mechanism by which cellulase- and xylanase-encoding genes are induced. The inducing abilities of various saccharides have been tested, and some saccharides induce the synthesis of both xylanases and cellulases (10, 21, 31, 37, 48). Nevertheless, on the basis of biochemical data (1, 20, 21) and mRNA expression analysis data (23, 31), a separate induction mechanism has been proposed for these systems in both Aspergillus and Trichoderma. Recently, a selection system was developed to isolate Aspergillus niger strains having mutations in a transcription factor involved in induction of expression of xylanolytic genes. Complementation of such a mutation by transformation with a plasmid library led to the isolation of the A. niger xlnR gene, which encodes a transcriptional activator of the A. niger xylanolytic system (44). This xlnR gene encodes a zinc binuclear cluster protein, which is a member of the GAL4 family of transcription factors. Isolation of both the xlnR gene and A. niger xlnR loss-of-function mutants provided an opportunity to study the spectrum of genes that are controlled by XlnR at the transcriptional level.