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Genome and proteomic analysis of industrial fungi

Genome and proteomic analysis of industrial fungi. Scott E. Baker Pacific Northwest National Laboratory BMS Annual Scientific Meeting: Exploitation of Fungi Manchester, UK September 6, 2005. Products: Fuels and chemicals. Products: Fuels and chemicals. Petroleum. Petroleum refinery.

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Genome and proteomic analysis of industrial fungi

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  1. Genome and proteomic analysis of industrial fungi Scott E. Baker Pacific Northwest National Laboratory BMS Annual Scientific Meeting: Exploitation of Fungi Manchester, UK September 6, 2005

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  3. Products: Fuels and chemicals Products: Fuels and chemicals Petroleum Petroleum refinery Complex biomass: Agricultural products and “waste” Bio-refinery Current andfutureroutes to fuels and chemicals Petroleum products Biobased products 3

  4. Products: Fuels and chemicals Bio-refinery Filamentous fungi inside the Bio-refinery Fungal fermentationand catalysis Fungal fermentationand catalysis Simple sugars Complex biomass: Agricultural products and “waste” 4

  5. The world of the mycologist… 5

  6. Publicly available and pending fungal genome sequence databases 6

  7. Why fungal genomics? • Genome sequence paints a high level picture of organism biology • Genome sequence is a platform for discovery • Genome sequence enables other high throughput discovery tools (proteomics, transcriptomics, etc) • A genome project can unite/revive a research community 7

  8. http://www.aspergillus.man.ac.uk/indexhome.htm?secure/sequence_info/index.php~mainhttp://www.aspergillus.man.ac.uk/indexhome.htm?secure/sequence_info/index.php~main 8

  9. Why a public Aspergillus niger genome project? • A bioprocess organism • First citric acid process reported in 1917 with wildtype ATCC 1015 Aspergillus niger strain • Highly efficient fermentation of glucose to citric acid • A protein production organism • Source of important enzymes • Industrial protein producer • Sequenced twice by industry • Public access to sequence with restriction • Large economic footprint 9

  10. ATCC 9029 NRRL 3122 10

  11. ATCC 9029 NRRL 3122 ~10X ~80X 11

  12. PNNL A. niger strain (sequenced by Integrated Genomics) Phylogeny from Robert A. Samson, Jos A.M.P. Houbraken, Angelina F.A. Kuijpers, J. Mick Frank and Jens C. Frisvad. 2004. New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Studies in Mycology. 50:45-61. 12

  13. The DOE Aspergillus niger genome project • Proposed to the US Department of Energy Microbial Genome Program by the PNNL Fungal Biotechnology team • Collaboration with DOE’s Joint Genome Institute • Current status • Final coverage: ~8X shotgun • Production sequenced to 4X and QC assembly performed, 8X sequencing to be completed by September 14th, assembly and automated annotation to follow • EST libraries constructed from RNA isolated from citric acid production and complex biomass digestion conditions~25,000 to be sequenced • Annotation: In collaboration with JGI/LANL • Public release: Target of December 1st • Other Aspergillus niger genomes • ATCC 9029: low coverage, sequenced by Integrated Genomics – Sequence available on request. Contact Scott Baker or Jon Magnuson (scott.baker@pnl.gov or jon.magnuson@pnl.gov) • CBS 513.88: DSM – announced public release at Asilomar FGC 13

  14. Integrated Genomics JGI • Date • Method • Coverage • Genomic library insert size • Contigs or scaffolds 2000 Shotgun No finishing ~4-6X 1-2kb >9000 contigs 2005 Shotgun Plus finishing ~8X 3kb 8kb 40kb <100 scaffolds 14

  15. The “QC” A. niger ATCC 1015 assembly – 4X coverage • total number of scaffolds: 118 • total length of scaffolds: 35634017 • N50 scaffold number: 6 • N50 scaffold size: 1931570 • total number of contigs: 1646 • total length of contigs: 34162656 • N50 contig number: 215 • N50 contig size: 47636 • Total: 243,688 reads • 3 kb: 105,065 = 43.1% • 8 kb: 118,655 = 48.7% • 40 kb: 19,968 = 8.2% 15

  16. The “QC” A. niger ATCC 1015 assembly – 4X coverage • Over 40 that encode ketosynthase and acyl-transferase domains(i.e. PKSs and FASs) • Mat-1-1(alpha box) • ~95% of the of the genome is found in 24 scaffolds – 1.5 scaffolds/chromosome arm • EST coverage/annotation • Genencor to release ~7500 EST sequences from several different growth condition libraries • JGI will sequence 25,000 ESTs • The Fungal Genomics program at Concordia University will contribute ~12,000 ESTs • Annotation jamboree tentatively scheduled for April 2006, following the European Conference on Fungal Genetics • Limited gap closure or “finishing” is planned by JGI-LANL 16

  17. What’s next? A multi-gene phylogeny and more genomes from Aspergillus section Nigri PNNL A. niger strain (sequenced by Integrated Genomics) DOE MGP Proposed July 14th Phylogeny from Robert A. Samson, Jos A.M.P. Houbraken, Angelina F.A. Kuijpers, J. Mick Frank and Jens C. Frisvad. 2004. New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Studies in Mycology. 50:45-61. 17

  18. Genome sequence…so what?

  19. General Procedure for Proteomics Lyse cells Digest with trypsin • Perform • LCQ • analysis Isolate proteins Separate in one or more dimensions reverse phase, ion exchange Run data through peptide identifying program (SEAQUEST) MS Raw Data Identify unique peptide = identify parent ORF MS/MS 19

  20. Quantitative proteomics • Used for comparison of biological samples generated by two or more different experimental conditions • Current technologies utilize isotopic labeling strategies • ICAT • Metabolic labeling • Pairwise comparison • Our goal: Generate a quantitative proteomic methodology using statistical analysis of raw MS abundance data and that does not use isotopic labeling 20

  21. MASIC: A program for measuring ion peak intensity and area 21

  22. There are lies, there are damn dirty lies and there are statistics… 22

  23. Peptides from sample protein 23

  24. Statistical analysis I 24

  25. Statistical analysis II 25

  26. Relative quantitation with confidence intervals (95%) 26

  27. “Global” proteomic summary chart 27

  28. Proteomics summary and future directions • Using statistical analysis of raw mass spec data we have developed a methodology for relative quantitation of proteins across multiple samples • Future experiments: • Time course experiment – Citric acid production in Aspergillus niger • Strain comparison – Trichoderma reesei QM6a vs Rut-C-30 • Other comparisons – “Production” strains vs. wildtype • Internal standards for greater quality control • Isotopic peptides for “absolute” quantitation 28

  29. Acknowledgements PNNL Fungal Biotechnology Ziyu Dai Jon Magnuson Chris Wend Ellen Panisko Ken Bruno Kyle Fowler Kelly Vincent Bob Romine Beth Hofstad Mark Butcher Katie Panther Dennis Stiles Linda Lasure PNNL Proteomics QC Analysis Team Don Daly Kevin Anderson Matt Monroe Phylogenetic analysis Rob Samson CBS Jens Frisvad DTU Dave Geiser Penn State DOE JGI A. niger ATCC 1015 genome Dan Drell Erika Lindquist Diego Martinez Paul Richardson Dan Rokhsar Chris Detter …the list continues to grow! David Bruce Secreteome analysis Adrian Tsang Concordia U 29

  30. Future genomes… Two “lower” fungi through the JGI CSP Piromyces Phycomyces (led by Luis Corrochano) Phycomyces fungi, showing sporangiophores (fruiting bodies) in the wild type and color mutants. Photo by Tamotsu Ootaki. Piromyces sp E2.. Photo by Johannes Hackstein. http://www.jgi.doe.gov/sequencing/why/CSP2006/piromyces.html http://www.jgi.doe.gov/sequencing/why/CSP2006/Pblakesleeanus.html 30

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