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
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
Model organism for eukaryotic biology • Circadian rhythms • Epigenetics • Genome defense • Mitochondrial biology • Post-transcriptional gene silencing • DNA repair • One gene, one enzyme hypothesis
Genome-scale metabolic models Minimal media Wild-type + Minimal media = Growth Essential compounds
Genome-scale metabolic models Minimal media Mutant + Minimal media = No Growth Essential Compounds
Genome-scale metabolic models Minimal media + supplement Mutant + Minimal media + Supplement = Growth Essential compounds
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
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
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
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
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.
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.
Modular biomass composition 893 reactions assigned to 257 Pathways DNA Amino acids Cell wall … Lipids Sterols Essential cofactors Secondary metabolites
Fast Automated Reconstruction of Metabolism (FARM) • Linear metabolite dilution FBA (limed-FBA) • One-step functional Pruning (OnePrune) • Consistent Reproduction of Phenotype (CROP)
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)
Literature curation confirms CROP suggestions 491 citations covering 47% of all enzyme catalyzed reactions …
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!
FBA doesn’t account for metabolite dilution, so it allows cycles that lack an input flux
limed-FBA accounts for metabolite dilution, so it disallows cycles that lack an input flux