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.
Director DSM Research and Development Life Sciences Joop Roels continues: "Since the DNA sequence of A. niger is almost 3 times larger than the bakers' yeast genome which is already known, the task for DSM and its partners was challenging. Ultimately, the sequencing of A. niger required the identification of over 13,000 genes encoded in approximately 34.5 million base pairs, and has been achieved in a remarkably short period."
Accelerate development
When DSM initiated the project in July 2000, only 1-2 % of the A. nigers
genes were known. Accuracy of the genetic sequence - critical to meaningful
interpretation of such complex data - has been ensured by 8-fold coverage
of the complete sequence and computer prediction/verification modeling. Knowledge
of A niger's DNA sequence allows a deeper understanding of its physiological
behavior and will accelerate the development of additional analytical tools
like proteomics. DNA arrays will now be designed which can be used to unravel
the complex relationships between processes in living cells at a molecular
level.
"By understanding the mechanisms of gene expression in certain enzyme production processes, it is possible to fine-tune individual enzyme activities - offering the possibility of tailoring products to customer's specific needs," continues Joop Roels.
"Our research and process development staff is confident of being able
to replace current trial-and-error procedures with a rational, genomic-based
approach. The result will be improved efficiency of a wide range of fermentation-based
production processes - leading to lower costs, and accelerated development
of new and improved products. We forecast significant benefits for our customers
in the bakery, fruit juice, beer, wine, dairy, nutraceutical and animal feed
industries."
"Completion of the DNA sequencing phase of the Aspergillus niger genomics project will generate new opportunities to improve and protect DSM's technology base," adds Wim de Boer, intellectual asset manager of DSM Life Science Products. "However, we understand that the genome could also be of significant interest to many other parties in industry and the academic world. DSM is therefore offering to make the results available to commercial partners within its existing licensing arrangements. In addition, a low-barrier access program will be made available to academic organizations from 1 January 2002."
The successful sequencing of A. niger builds on DSM's
involvement in previous genomics programs, including the sequencing of the
Bacillus subtilis and the bakers yeast genomes and in establishing the DNA-sequence
of the bacterium Propionibacterium freudenreichii, a bacterium used in the
dairy industry.
DSM's partners in the A. niger genomics project include the German
companies Gene Alliance (high-throughput DNA sequencing) and Biomax (bio-informatics),
the University of Amsterdam (DNA micro-arrays) and the University of Utrecht
(proteomics).
Exploring and exploiting the Aspergillus
niger genome.
Gert Groot *, Herman Pel, Noël van Peij, and Albert van Ooyen, DSM Food
Specialties R&D, P.O. Box 1, 2600 MA Delft, The Netherlands.
The filamentous fungus Aspergillus niger is the main microorganism
used for large scale fermentative production of enzymes within DSM. In order
to rationalize the ongoing improvement of production strains and processes,
as well as to speed up the identification of potential new enzyme products,
DSM recently completed the sequencing and annotation of the A. niger genome.
The sequencing project consisted of the 7.5x coverage shotgun sequencing of
over 500 carefully selected genomic BAC clones. The 34.5 Mb of unique sequence
obtained could be assembled into 19 supercontigs that represent the eight
A. niger chromosomes. Genome statistics such as gene density, intron distribution
and the presence of transposons will be presented.
Using computer-assisted manual gene modeling and annotation over 14,000 orfs
were identified and functionally classified. A strong functional prediction
could be made for approx. 45% of the orfs. EC numbers as well as many other
functional classifications were assigned to over 3,000 genes. A first inventory
of the protein functions identified revealed large numbers of previously unknown
enzymes with potential industrial relevance. The availability of the genomic
sequence allows a detailed study of the regulatory sequences of these genes.
The functional annotation of genes revealed a detailed blueprint of for example
primary metabolism, intracellular transport and protein secretion. The availability
of the complete genome allows an in-depth comparison of cellular processes
in a variety of physiological conditions and in different strains using modern
high throughput techniques such as transcriptomics and proteomics.
The Aspergillus niger genome was sequenced by Gene
Alliance.The genome was annotated by Biomax Informatics.
Geleen, The Netherlands and Hilden, Germany, September 15th , 2000 – DSM Research,a division of DSM N.V. (Amsterdam: DSM.ASX), has commissioned the German genomics consortium Gene Alliance to determine the DNA sequence of the fungus Aspergillus niger, one of the most important organisms used in enzyme production at DSM.
The DNA sequence of Aspergillus niger is estimated to consist of approximately 30 million base pairs, which would make this study what is believed to be the largest industrial genome contract sequencing project in Europe to date. Since DSM expects to be able to use the results of the project to substantially improve several of its biotechnological processes over the next few years, it considers the study to be very important for reinforcing its competitive position in the life sciences field. The DNA sequencing of Aspergillus niger is scheduled to be completed in September 2001. The project will be conducted for DSM on a fee for service basis by the Gene Alliance. Financial terms were not disclosed.
The high-throughput sequencing of the Aspergillus niger genome will be conducted by the members of the Gene Alliance (AGOWA (Berlin), Biomax Informatics (Munich), GATC (Konstanz), MediGenomix (Munich), and QIAGEN GmbH (Hilden))and coordinated by QIAGEN. Each of the Gene Alliance partners' capacity will be optimally leveraged. The project will employ a BAC by BAC sequencing approach. The bioinformatics aspects of the project such as full annotation and DNA/protein visualization, which are required to establish what genes are present and what their possible functions are, will be carried out by Biomax Informatics, also a Gene Alliance member, using the GeneRelianceä bioinformatics system. The GeneReliance system is based on the Pedant-Proä Sequence Analysis software developed at Biomax. The system identifies all genetic elements within DNA sequences and assigns functional classification of all open reading frames (ORFs) which will give DSM full use of the data.