DOE Genome Sequencing Nomination
Nomination for additional genome sequence coverage of Aspergillus niger
Aspergillus niger group:
Scott Baker, Jon Magnuson, Ziyu Dai, Ellen Panisko, Chris Wend and Linda Lasure, Pacific Northwest National Laboratory
Point of Contact/PI:
Pacific Northwest National Laboratory
902 Battelle Blvd.
P.O. Box 999
Richland , WA 99352
Phone: 509-372-4759
FAX: 509-372-4732
Nomination for additional genome sequence coverage of Aspergillus niger
Summary
1. Relevance to DOE missions
Citric acid production by the filamentous ascomycete fungus, Aspergillus niger probably represents the most efficient, highest yielding bioprocess in practice. This process is a model for other filamentous fungal fermentation processes that will become a key part of DOE's vision of the biorefinery, where multiple products, such as, organic acids and ethanol, are produced from renewable biomass. These products can be further refined for use as plastic monomers, solvents, or fuels, thereby decreasing dependence on petroleum, the traditional source of these products. Indeed, while much is known about the fermentation process for citric acid production, relatively little is known about the basic science of A. niger , whether in its natural role as a soil fungus or in its role as an industrial organism for the production of organic acids and enzymes.
As a common member of the microbial communities found in soils, A. niger also plays a significant role in the global carbon cycle. It is a soil saprobe with a wide array of hydrolytic and oxidative enzymes involved in the breakdown of plant lignocellulose. An increased understanding of the molecular mechanisms controlling carbon flux in fungi will be gained from study of the A. niger genome. Indeed, as more fungal genomes are sequenced, scientists will be able to use comparative genomics to find commonalities and differences in the molecular circuits that control carbon flux in fungi.
A. niger is an important model fungus for the study of eukaryotic protein secretion in general, the effects of various environmental factors on suppressing or triggering the export of various biomass degrading enzymes, molecular mechanisms critical to fermentation process development, and mechanisms involved in the control of fungal morphology. High quality genomic sequence will enable the application of high throughput global proteomics, reverse genetic studies of genes, and a variety of bioinformatic analyses, to basic and applied research problems in this microorganism. The research issues of interest and methods for studying these issues are compatible with goals of the DOE Genomes:GTL program. The generation of a high quality publicly accessible A. niger genome sequence will be extremely valuable to several scientific disciplines, both basic and applied.
2. Suitability for whole genome shotgun sequencing
The approximately 32Mb genome of Aspergillus niger , ATCC strain 9029, was sequenced to a depth of ~4-6× coverage by Integrated Genomics, Inc. in 2001. In 2004, this sequence was purchased by Pacific Northwest National Laboratory with funds from the LDRD Biobased Products Initiative. Integrated Genomics used genomic DNA that was mechanically fragmented to ~2kb before insertion into vectors for sequencing. In June of 2004, JGI used their Jazz assembler to align the available sequencing reads resulting in a large number of scaffolds that correlated with the expected genome size of A. niger . This was encouraging, but this initial assembly also revealed that the quality of the genome sequence could be greatly improved by an additional 2× of sequence coverage using larger genomic fragments to fill in significant gaps in the scaffolds.
3. Source for genomic DNA
Genomic DNA will be prepared from Aspergillus niger ATCC strain 9029 by scientists in the Fungal Biotechnology group at Pacific Northwest National Laboratory who are experienced in the care and handling A. niger and all of its biomolecules: DNA, RNA, proteins and metabolites.
4. Post-sequencing work
Upon assembly of the sequencing reads, data will be released via the JGI website and genome browser to the scientific community. We plan to use the automated annotation pipeline established at JGI for the creation of gene models. Gene models will be released to the public (via the JGI website/genome browser) immediately.
We also intend to manually annotate a number of gene families relevant to A. niger biology. Annotation of genes involved in 1) primary metabolism will be led by Dr. Magnuson, 2) secondary metabolism will be led by Dr. Baker, 3) complex organic substrate breakdown will be led by Drs. Magnuson and Baker and 4) morphology control and transcriptional regulation will be led by Dr. Dai. Manual annotations will be added to the JGI website as they are completed. We expect that the A. niger genome will attract considerable scientific interest and intend to invite all interested researchers to join in the annotation, in order to make it a community effort.
Annotation and genomic sequence will be deposited into NCBI GenBank concurrent with completion of manual annotation efforts.
5. Availability of molecular genetic analytical tools
Aspergillus niger is genetically tractable. A number of methodologies for both targeted and random gene transformation have been in use for several years. These include Agrobacterium tumefaciens mediated transformation which has been extremely efficient in our experience, and protoplast preparation followed by polyethylene glycol stimulated DNA uptake.
6. Biological novelty/uniqueness
Contributing to the biological novelty and uniqueness of A. niger is that research on this organism is of high interest to several scientific and engineering disciplines for both applied and basic research.
Citric acid production using Aspergillus niger has been practiced in one form or another since 1917 and represents the most efficient filamentous fungal fermentation process ever developed (Currie, 1917). A. niger is the model fungus in which to study what many consider the model fermentation process. While the citric acid process has been studied and optimized for decades, the lack of an available genome sequence has made global “systems biology” studies impossible. The molecular mechanisms controlling fungal morphology are of interest to scientists studying human health (fungal dimorphism and pathology) and to scientists interested in developing highly efficient processes for the biorefinery. In regard to the latter, the issue is filamentous versus pelleted (< 0.5 mm dia.) growth of fungi in submerged culture. Pelleted growth is highly correlated with high production rates and final product yields, due in part to more efficient mass transfer with regard to substrates, oxygen and product (Dai et al., 2004).
A. niger is also a model organism for eukaryotic protein processing and secretion. A niger is often used for overexpression of native and heterologous proteins, and has been the subject of research involving the expression of glycoproteins with human-like glycosylation (Ngiam et al., 2000; Derkx et al., 2001; Valkonen et al., 2003; Mulder et al., 2004; Ward et al., 2004). In order to better understand the limitations to overexpression of heterologous proteins, the unfolded-protein response of A. niger has been studied extensively. Among the proteins of interest that are secreted by A. niger , plant cell wall degrading enzymes are of particular interest; indeed, a variety of glycosyl hydrolases are commercially produced using A. niger . Thus, this organism is an ideal model for the study of complex carbohydrate degradation as well. Various glycosyl hydrolases are critical to the conversion of lignocellulosic biomass to sugars for the biorefinery of the future.
7. The unique position in the “Tree of Life”
Aspergillus niger is a filamentous ascomycete fungus from the order Eurotiales and family Trichocomaceae which includes all Aspergillus and Penicillium species . The genus Aspergillus is divided into three sub-genera, Aspergillus , Nidulantes and Fumigati . These subgenera are further divided into several sections. A. niger resides in subgenus Aspergillus section Nigri (Fig. 1). Aspergillus is now becoming the genus of choice for large scale comparative evolutionary studies. As multiple Aspergilli genomes become sequenced, there is an opportunity for comparative studies using whole genome alignments. Unfortunately, the sequenced Aspergilli, A. fumigatus , A. nidulans and A. oryzae (sequenced but not released) are too distantly related to give much useful information about conserved regulatory motifs, speciation and recent genomic rearrangements. However with the impending sequencing of the A. flavus genome ( Gary Payne , personal communication), the planned release of A. oryzae and the prospect of a high quality draft of A. niger , the prospect of a detailed comparative evolutionary study of a filamentous fungus is very realistic. A similar study generated a large amount of novel information about genes and regulatory elements in budding yeast (Kellis et al., 2003; Cliften et al., 2003; Dujon et al., 2004).
8. Scientific impact
As mentioned previously, the potential impact to the scientific community is large. A. niger is an important organism for both basic and applied scientific research. Currently, there are no available genomic sequences for this particular clade of Aspergillus species.
Additional scientific impact would be felt in the Aspergillus specific research community as A. niger will be useful in the comparative studies with A. fumigatus, A. oryzae (sequenced, but not released) and A. nidulans .
9. Description of the user community and resources
The Aspergillus user community is large and organized. Recently, the community formed the Aspergillus Genomes Research Policy Committee to coordinate research efforts across all labs working on Aspergillus species (see attached letter of support). With this committee in place, there is an infrastructure to use available genomes for large scale comparison, to help in manual annotation efforts, to pool resources for common research interests, such as DNA microarrays, and to form collaborations in areas such as proteomics and metabolomics.
10. Commitment to data release schedule
We are absolutely committed the DOE's data release schedule.
References
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