4.2.1.1 Fungal Genomics

1. 4.2.1.1 Fungal Genomics Biochemical and Products Peer Review Scott E. Baker Pacific Northwest National Laboratory August 8, 2007

2. 4.2.1.1 Fungal Genomics Overview Start: January 2005 End: December 2012 Pt-C. Chemical and Materials Organism Development Bt-F. Cellulase Enzyme Production Bt-J. Fuels Organism Development Recommended time for this slide: <2 min The purpose of this slide is to provide some context for evaluating your project and especially your accomplishments. The information on the left describes the magnitude and timing of the investment in your project. The information on the right describes the players and the issues that are to be overcome. For projects that include multiple partners, please discuss the roles of each and how the overall project is being managed. Include stage from stage gate guide.Recommended time for this slide: <2 min The purpose of this slide is to provide some context for evaluating your project and especially your accomplishments. The information on the left describes the magnitude and timing of the investment in your project. The information on the right describes the players and the issues that are to be overcome. For projects that include multiple partners, please discuss the roles of each and how the overall project is being managed. Include stage from stage gate guide.

3. Goals and Objectives Goal Generate innovative fungal-based biotechnology to enable a robust biorefinery industry Objectives Reduce the cost of biofuels and bioproducts Directly utilize complex biomass Enable processes with high concentration of end-product (for example, organic acids and enzymes) Improve the overall tractability of filamentous fungi Leverage industry needs and expertise to help guide research program

4. Background & OBP Relevance

5. Genesis of the Fungal Genomics project Chemical building block products Morphology control genes Filamentous fungal ethanol Fusarium oxysporum � 5.8% (w/w) on 10% glucose + 0.2% DDG Rationale behind the Fungal Genomics project Filamentous fungal bioprocesses are among the most productive known Filamentous fungi are foundational organisms for the biorefinery Co-products improve ethanol (and other renewable fuel) economics Background & OBP Relevance

6. Targets for products 40-50g/L organic acid baseline fermentation Ability to genetically manipulate organism Improvement to 60-80g/L in 12-24 months OBP Milestones M 1.2 Demonstrate and validate economical fiber conversion to C5 and/or mixed C5/C6 sugars in a wet mill by 2007 M 1.3 Demonstrate and validate economical conversion of mixed sugars to ethanol M 1.4 Demonstrate and validate economical new products from C5 or mixed C5/C6 sugars in a wet mill by 2008 M 1.5 Demonstrate and validate economical new products from C6 sugars in a wet mill by 2008 Background & OBP Relevance

7. Targets for filamentous fungal ethanol 3-6% (w/w) ethanol final product from glucose and/or xylose fermentation Ethanol production directly from biomass Results in decreased enzyme cost OBP Milestones M.4.5.5: By 2012, validate integrated pretreatment and enzymatic hydrolysis of corn stover (dry and wet) at pilot scale M.4.6.3: By 2012, validate/optimize integrated production of ethanol from cornstover derived sugars at pilot scale M.5.4.5: By 2017, validate integrated pretreatment and enzymatic hydrolysis of switchgrass at pilot scale M.5.6.3: By 2017, validate integrated production of ethanol from switchgrass derived sugars at pilot scale Background & OBP Relevance

8. Scientific Approach

10. Scientific Approach Fungal Genomics Core Tasks Morphology control Genomics and proteomics Hyperproductivity Biomass to product Partner Review Board Filamentous fungal ethanol Include here higher level tasks and funding for the entirety of the project by Fiscal YearInclude here higher level tasks and funding for the entirety of the project by Fiscal Year

11. Scientific Approach

12. Citric acid production by Aspergillus niger : A model fungal fermentation system

13. Citric acid production by Aspergillus niger : A model fungal fermentation system

14. Accomplishments: Task 1Morphology

15. Accomplishments: Task 1Morphology

16. Large amounts of reactive oxygen species (ROS) in Aspergillus niger pellets

17. Accomplishments: Task 1

18. Proposed to the US DOE Microbial Genome Program by the PNNL Fungal Biotechnology team Genome project funded by DOE SC OBER (direct to JGI) Collaboration with DOE�s Joint Genome Institute Current status Final draft coverage: ~7.88X shotgun EST libraries constructed from RNA isolated from citric acid production and complex biomass digestion conditions~30,000 sequenced Annotation: In collaboration with JGI/LANL Gap closure � by Stanford-JGI 454 sequencing � eight runs Public release: April 2006 Anticipated publication winter 2007

19. Aspergillus niger genome map

20. Goal to sequence 30-50 fungal genomes over 3-5 years Whitepaper completed June 2006 Steering committee with representatives from seven DOE labs (LBNL, LLNL, ORNL, LANL, PNNL, ANL, NREL), academia and industry Condensed version to be published as a Commentary in a peer reviewed journal (Fungal Biology Reviews) List of organisms picked based on relevance to DOE Bioenergy Mission Top six organisms entering sequencing queue in FY07 Accomplishments: Task 2Genomics

22. Accomplishments: Task 5Partners Review Board Partner Review Board established Poet Novozymes Verenium Dyadic International Mycosynthetix 2005, 2006 and 2007 Annual Partner Meetings 2006 and 2007 Annual Report to Partner Board SharePoint Collaboration Site launched

23. Accomplishments: Task 5Partner Review Board

24. Accomplishments: Task 6Filamentous fungal ethanol Preliminary data Fusarium oxysporum � 5.8% (w/w) on 10% glucose + 0.2% DDG Task being added for FY08 Metabolic modeling Targeted screening

25. Success Factors and Showstoppers Targets for products 40-50g/L organic acid baseline fermentation Ability to genetically manipulate organism Improvement to 60-80g/L in 12-24 months Targets for filamentous fungal ethanol 3-6% (w/w) ethanol final product from glucose and/or xylose fermentation Ethanol production directly from biomass Results in decreased enzyme cost

26. Success Factors and Showstoppers Showstoppers Intractable biological hurdle Not economically viable process Competitive technology Window of opportunity Development of toolset with product application: 4-10 years Adoption within biorefinery for products and fuels: 7-15 years

27. Future Work Future research Task 1 (Morphology) Refine and test Morphology v1.0 model Concentrate on ubiquitin and ROS Task 2 (Genomics) Complete A. niger genome analysis Proteomic characterization of pellet vs. filamentous Transcriptome analysis collaboration with Jen Nielsen (DTU) Tasks 3 & 4 (Hyperproductivity & Biomass to Bioproduct) Baseline A. terreus itaconic process on biomass Test morphology genes in A. terreus Economic & process modeling Task 5 (Partner Review Board) Update PRB SharePoint FY08 Annual Report Task 6 (Filamentous Fungal Ethanol) Implement metabolic flux modeling with ethanol production as a target product Targeted screening of Mycosynthetix culture collection Economic & process modeling Recommended time for this slide: 3 min Purpose: Describe what will be done in the remainder of this year and in the coming year.Recommended time for this slide: 3 min Purpose: Describe what will be done in the remainder of this year and in the coming year.

28. Future Work Key milestones Delivery of Morphology v1.0 Model (Sept 07) Complete A. niger genome analysis (Dec 07) Implement fungal metabolic flux modeling (June 08) Decision points & issues Frequent interaction with Partner Review Board to keep research relevant to biorefinery end-users Implement and use economic/process models Recommended time for this slide: 3 min Purpose: Describe what will be done in the remainder of this year and in the coming year.Recommended time for this slide: 3 min Purpose: Describe what will be done in the remainder of this year and in the coming year.

29. Summary Accomplishments Significant progress in understanding control of morphology in filamentous fungi Collaboration with DOE Joint Genome Institute to increase available fungal genome resources Establishment and interaction with industry through Partner Review Board Continued development of biotechnology toolsets to support OBP milestones Future Addition of filamentous fungal ethanol task to project Addition of metabolic flux modeling to capability set

30. PNNL Fungal Team

31. Responses to Previous Reviewers� Comments �Critical to have the right gene targets � this vulnerability not clear � could use a genome partner� We have succeeded in partnering with the DOE Joint Genome Institute on a number of fungal projects included the Aspergillus niger genome and a large fungal genome sequencing initiative �Are current commercial producers using FF [filamentous fungi] involved?� Yes, four of the five partners currently use filamentous fungal processes. �Are there other approaches other than proteomics, a current topic in favor?� We utilize a number of approaches: genetics, genomics, transcriptomics, proteomics, metabolic modeling, targeted screening

32. Publications and Presentations Publications Baker, SE. 2006. Aspergillus niger genomics: Past, present and into the future. Medical Mycology. 44:S17-21. Baker, S.E. and Bennett, J.W. An overview of the genus Aspergillus. �In The Aspergilli: Genomics, Medicine, Biotechnology and Research Methods.� Ed. G.H. Goldman and S. Osmani. In press. Baker, S.E., Wend CF, Martinez, D, Magnuson, JK, Panisko, EA, Dai, Z, Bruno, KS, Anderson, KK, Monroe, ME, Daly, DS, Lasure, LL. Genome and Proteome Analysis of Industrial Fungi. In "Exploitation of Fungi." Ed. G.D. Robson, P. van West, G.M. Gadd. Cambridge press. In press. Zhou, K, Panisko, EA, Magnuson, JK, Baker, S.E., Grigoriev, I. Proteomics for validation of automated gene model predictions. In �Mass Spectrometry of Proteins and Peptides� Eds. M. Lipton, L. Pa�a-Tolic. Humana Press. In press. Dai Z, KS Panther, LJ Hubbard, KS Bruno, JK Magnuson, SE Baker, and LL Lasure.� 2007.� "The G protein beta subunit regulates spore germination, vegetative growth and asexual sporulation of Aspergillus niger in response to nitrogen sources." In revision.

33. Publications and Presentations Invited Presentations Los Alamos Nat�l Lab Genome Explorer Seminar Series (2005, 2007) Society for Industrial Microbiology Annual Meeting (2005, 2006, 2007) Fungal Genetics Conference (2005, 2007) International Aspergillus Meeting (2005, 2006, 2007) British Mycological Society Annual Meeting: Exploitation of Fungi (2005) European Conference on Fungal Genetics (2006) Proctor & Gamble Conference on Innovation for Sustainability and Conservation (2007) NIChE (New Industrial Chemistry and Engineering) Conference (2007) World Congress on Industrial Biotechnology and Bioprocessing (2007) BIO (2007)