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

2

3 Current and future routes to fuels and chemicals Petroleum refinery Products: Fuels and chemicals Bio-refinery Complex biomass: Agricultural products and waste Products: Fuels and chemicals Petroleum products Biobased products

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

5 The world of the mycologist…

6 Publicly available and pending fungal genome sequence databases

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

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

10 ATCC 9029NRRL 3122

11 ATCC 9029NRRL 3122 ~80X ~10X

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 New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Studies in Mycology. 50:45-61.

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 DOEs Joint Genome Institute Current status Final coverage: ~8X shotgun Production sequenced to 4X and QC assembly performed, 8X sequencing to be completed by September 14 th, 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 or CBS : DSM – announced public release at Asilomar FGC

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

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

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

17 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 New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Studies in Mycology. 50: DOE MGP Proposed July 14th Whats next? A multi-gene phylogeny and more genomes from Aspergillus section Nigri

Genome sequence…so what?

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

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

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

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

23 Peptides from sample protein

24 Statistical analysis I

25 Statistical analysis II

26 Relative quantitation with confidence intervals (95%)

27 Global proteomic summary chart

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

29 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 Acknowledgements DOE JGI A. niger ATCC 1015 genome Dan DrellErika Lindquist Diego MartinezPaul Richardson Dan Rokhsar Chris Detter…the list continues to grow! David Bruce Phylogenetic analysis Rob SamsonCBS Jens Frisvad DTU Dave GeiserPenn State Secreteome analysis Adrian TsangConcordia U PNNL Proteomics QC Analysis Team Don Daly Kevin Anderson Matt Monroe

30 Phycomyces fungi, showing sporangiophores (fruiting bodies) in the wild type and color mutants. Photo by Tamotsu Ootaki. Piromyces sp E2.. Photo by Johannes Hackstein. Future genomes… Two lower fungi through the JGI CSP Phycomyces (led by Luis Corrochano) Piromyces