ABSTRACT
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.
Subject(s)
Aspergillus niger/genetics , Computational Biology/methods , Evolution, Molecular , Genetic Variation , Genome, Fungal/genetics , Phylogeny , Base Sequence , Gene Expression Profiling , Gene Rearrangement/genetics , Gene Transfer, Horizontal/genetics , Genomics/methods , Molecular Sequence Data , Polymorphism, Single Nucleotide/genetics , Sequence Analysis, DNA , Species Specificity , Synteny/geneticsABSTRACT
The ability to resist anti-microbial compounds is of key evolutionary benefit to microorganisms. Aspergillus niger has previously been shown to require the activity of a phenylacrylic acid decarboxylase (encoded by padA1) for the decarboxylation of the weak-acid preservative sorbic acid (2,4-hexadienoic acid) to 1,3-pentadiene. It is now shown that this decarboxylation process also requires the activity of a putative 4-hydroxybenzoic acid (3-octaprenyl-4-hydroxybenzoic acid) decarboxylase, encoded by a gene termed ohbA1, and a putative transcription factor, sorbic acid decarboxylase regulator, encoded by sdrA. The padA1,ohbA1 and sdrA genes are in close proximity to each other on chromosome 6 in the A. niger genome and further bioinformatic analysis revealed conserved synteny at this locus in several Aspergillus species and other ascomycete fungi indicating clustering of metabolic function. This cluster is absent from the genomes of A. fumigatus and A. clavatus and, as a consequence, neither species is capable of decarboxylating sorbic acid.
Subject(s)
Antifungal Agents/metabolism , Aspergillus niger/metabolism , Genes, Fungal , Metabolic Networks and Pathways/genetics , Sorbic Acid/metabolism , Alkadienes/metabolism , Amino Acid Sequence , Carboxy-Lyases/genetics , Decarboxylation , Fungal Proteins/genetics , Gene Order , Microbial Sensitivity Tests , Molecular Sequence Data , Multigene Family , Parabens/metabolism , Pentanes/metabolism , Sequence Alignment , Synteny , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Aspergillus niger/genetics , Aspergillus niger/metabolism , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Genomics , Industrial Microbiology , Gene Expression Profiling , Glucuronidase/biosynthesis , Glucuronidase/genetics , Proteome/analysisABSTRACT
Fungal mycelia are exposed to heterogenic substrates. The substrate in the central part of the colony has been (partly) degraded, whereas it is still unexplored at the periphery of the mycelium. We here assessed whether substrate heterogeneity is a main determinant of spatial gene expression in colonies of Aspergillus niger. This question was addressed by analyzing whole-genome gene expression in five concentric zones of 7-day-old maltose- and xylose-grown colonies. Expression profiles at the periphery and the center were clearly different. More than 25% of the active genes showed twofold differences in expression between the inner and outermost zones of the colony. Moreover, 9% of the genes were expressed in only one of the five concentric zones, showing that a considerable part of the genome is active in a restricted part of the colony only. Statistical analysis of expression profiles of colonies that had either been or not been transferred to fresh xylose-containing medium showed that differential expression in a colony is due to the heterogeneity of the medium (e.g., genes involved in secretion, genes encoding proteases, and genes involved in xylose metabolism) as well as to medium-independent mechanisms (e.g., genes involved in nitrate metabolism and genes involved in cell wall synthesis and modification). Thus, we conclude that the mycelia of 7-day-old colonies of A. niger are highly differentiated. This conclusion is also indicated by the fact that distinct zones of the colony grow and secrete proteins, even after transfer to fresh medium.
Subject(s)
Aspergillus niger/metabolism , Gene Expression Regulation, Fungal , Mycelium/metabolism , Cell Wall/metabolism , Fungal Proteins/metabolism , Genes, Fungal , Genome, Fungal , Glucans/chemistry , Maltose/chemistry , Models, Biological , Oligonucleotide Array Sequence Analysis , RNA, Fungal , Time Factors , Trans-Activators/metabolism , Xylose/chemistryABSTRACT
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.