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Lessons learned from the Genome-scale metabolic reconstruction and curation of Neurosporacrassa Jeremy Zucker Jonathan Dreyfuss Heather Hood James Galagan The Eli and Edythe L. Broad Institute A Collaboration of Massachusetts Institute of Technology, Harvard University and affiliated Hospitals, and Whitehead Institute for Biomedical Research
Capture Metabolic Knowledge Pathway-tools/BioCyc KEGG • Reactions • Interactions • Literature
Visualizing ‘omics Data Provide a visually intuitive, metabolic framework for interpreting large ‘omics datasets
in silico Predictions AlgorithmicallyInterpret Expression Data in a Metabolic Context?
Example: Plasmodium Eflux* • Validation • KO Phenotype Predictions – 90% Accuracy • External Metabolite Changes – 70% Accuracy • New Predictions • 40 Enzymatic drug targets • Experimental validation of novel target *Colijn, C., A. Brandes, J. Zucker, et al. (2009). PLoSComputBiol
Modeling in the Neurospora PO1 Clock Profiling Visualization and Analysis RNA-Seq ChIP-Seq Interpretation of Expression Profiling and Regulatory Network Data in a Metabolic Context – Inform Experiments
Manual reconstruction protocol Nature Protocols, Vol. 5, No. 1. (07 January 2010), pp. 93-121.
Automated Model SEED reconstruction pipeline Nature biotechnology, Vol. 28, No. 9. (29 September 2010), pp. 977-982
Genome sequence to metabolic model Elements Pathways Literature Metadata Nutrient media (Vogels) Complexes Reactions NeurosporaCyc Biomass composition Transporters
EFICAz2 predicts enzymes Databases 1993 enzymes 1770 reactions 9934 protein sequences HMMs FDR … Decision tree SVM BMC Bioinformatics 2009, 10:107
Protein Complex editor 31 complexes experimentally validated through literature search 182 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
Transport inference parser (TIP) 176 transporters assigned to 97 transport reactions 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.
Pathologic predicts pathways 1770 enzyme- catalyzed reactions 265 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.
Literature curation validates predictions 1212 citations associated with 307 pathways 31 complexes 168 genes … …
Solution: Instantiate polymerization steps • POLYMER-INST-Fatty-Acids-C16 + coenzyme A + ATP -> POLYMER-INST-Saturated-Fatty-Acyl-CoA-C16 + diphosphate + AMP + H+ • POLYMER-INST-Fatty-Acids-C14 + coenzyme A + ATP -> POLYMER-INST-Saturated-Fatty-Acyl-CoA-C14 + diphosphate + AMP + H+ • … • POLYMER-INST-Fatty-Acids-C0 + coenzyme A + ATP -> POLYMER-INST-Saturated-Fatty-Acyl-CoA-C0 + diphosphate + AMP + H+
What happens when the metabolic network is infeasible? • Add a “reaction” with the smallest number of reactants and products that results in a feasible model minimize card(r) subject to Sv + r = 0 l ≤ v ≤ u
Fast Automated Reconstruction of Metabolism • Input: • EFICAz probabilities for each reaction • Biomass components • Experimental growth / no growth phenotypes in different nutrient conditions • Gene essentiality • Manual curation of pathways • Output: • Metabolic network of MetaCyc reactions maximally consistent with input
Neurospora phenotypes for validation Neurosporae-Compendium 29 Mutants essential on minimal media Non-essential on supplemental media PO1 Phenotype Collection 79 non-essential KOs under minimal media Additional phenotypes are observed. Used FBA with Neurospora model to simulate gene knockouts in minimal medium
Comparison of model organisms under minimal media  Genome Res. 2004. 14: 1298-1309  Molecular Systems Biology 2007 3:121
Modeling the effect of oxygen limitation on xylose fermentation
Biofuels from Neurospora? • Growing interest for obtaining biofuels from fungi • Neurosporacrassahas more cellulytic enzymes than Trichodermareesei • N. crassacan degrade cellulose and hemicellulose to ethanol [Rao83] • Simultaneous saccharification and fermentation means that N. crassais a possible candidate for consolidated bioprocessing Xylose Ethanol