Using a genome-scale metabolic model of Neurospora crassa

1. Using a genome-scale metabolic model of Neurosporacrassa to capture genotype-phenotype relationships in an executable form Jeremy Zucker Broad Institute of MIT and Harvard Boston University

2. Outline • Importance of Neurospora to the history of predicting cellular metabolism and phenotypes • From annotated genomes to metabolic models using pathway and phenotype-directed curation • Model validation • Biological insights • Biolog analysis

3. Contributions of the lowly bread mold Neurosporacrassa

4. Model organism for eukaryotic biology • Circadian rhythms • Epigenetics • Genome defense • Mitochondrial biology • Post-transcriptional gene silencing • DNA repair • One gene, one enzyme hypothesis

5. Cellular metabolism and phenotypes

8. Genome-scale metabolic models Minimal media Wild-type + Minimal media = Growth Essential compounds

9. Genome-scale metabolic models Minimal media Mutant + Minimal media = No Growth Essential Compounds

10. Genome-scale metabolic models Minimal media + supplement Mutant + Minimal media + Supplement = Growth Essential compounds

11. Systems Biology of Neurospora Clock Profiling Visualization and Analysis RNA-Seq ChIP-Seq Interpretation of Expression Profiling and Regulatory Network Data in a Metabolic Context – Inform Experiments

12. From annotated genomes to metabolic fluX models

14. Pathway-directed curation

15. Enzyme function prediction from sequence Databases 808 genes assigned to 1023 enzyme activities 9934 protein sequences HMMs FDR … Decision tree SVM BMC Bioinformatics 2009, 10:107

16. Reaction direction determined by Gibbs free energy Group contribution method 1031 irreversible reactions 328 reversible reactions 673 chemical structures Biophys J 2008 Aug:95(3) 1487-99

17. Protein Complex editor 47 complexes experimentally validated through literature search 291 reactions with isozymes or complexes Identify multiple genes of reaction Present all possible combinations of complexes 2-oxoisovalerate complex • 2-oxoisovalerate alpha subunit • 2-oxoisovalerate beta subunit • … • fatty acid synthase beta • subunit dehydratase • fatty acid synthase alpha • subunit reductase … Allow curator to validate potential complexes Fatty acid synthase complex

18. Transport inference parser (TIP) 290 transport reactions 137 metabolites exported or transported between cytosol and organelles Filter proteins for transporters 9934 free-text Protein annotations Infer multimeric complex • MFS glucose transporter • ATP synthase • … • sucrose transporter Infer substrate … Infer energy-coupling mechanism Bioinformatics (2008) 24 (13): i259-i267.

19. Pathologic predicts pathways 1770 enzyme- catalyzed reactions 893 reactions assigned to 257 Pathways X = #rxns in metacycpwy … Y = #rxns with enzyme evidence … Z = #unique rxns in pwy P(X|Y|Z) = prob of pwy in Neurospora Science 293:2040-4, 2001.

20. Modular biomass composition 893 reactions assigned to 257 Pathways DNA Amino acids Cell wall … Lipids Sterols Essential cofactors Secondary metabolites

21. Initial draft of Neurospora model

22. Phenotype directed curation

23. Fast Automated Reconstruction of Metabolism (FARM) • Linear metabolite dilution FBA (limed-FBA) • One-step functional Pruning (OnePrune) • Consistent Reproduction of Phenotype (CROP)

24. Consistent Reproduction of Phenotype(CROP) • Input: • Experimental evidence for reaction • Pathway evidence for reaction • Thermodynamic estimate of Gibbs free energy • Probabilistic evidence of enzyme function • Training set of growth/no-growth mutant phenotypes • Output: • Suggests reactions to remove (MILP) • Suggests reactions to add (linear relaxation of MILP)

25. Literature curation confirms CROP suggestions 491 citations covering 47% of all enzyme catalyzed reactions …

26. Model validation

27. Essential gene training set accuracy

28. Essential gene test set accuracy

29. Auxotroph rescue

30. Embracing our outliers • Δace-2,3,4 can grow in acetate minimal media, but not sucrose minimal media, even though sucrose can be converted to acetate! • gln-1 and gln-2 are both required for glutamine synthase—sometimes! • Δerg-14 is predicted lethal, but the knockout mutant grows!

31. Mechanistic insights and testable hypotheses

33. FBA doesn’t account for metabolite dilution, so it allows cycles that lack an input flux

34. limed-FBA accounts for metabolite dilution, so it disallows cycles that lack an input flux

35. limed-FBA correctly predicts arg-14 essentiality, but FBA does not

36. Comparison to other constraint-based methods

37. Effect of oxygen limitation on xylose fermentation

42. Biolog analysis

43. Negative control well at 0h

44. Negative control well at 72h

45. Negative control experiments

46. Biolog prediction accuracy

48. Carbon sources

49. Nitrogen sources

50. Sulfur sources