Figure 1.
The process used for the reconstruction and validation of the metabolic model is described in four stages. In the first stage, pathway-directed curation, the genome sequence annotation [14], [15] , metabolic pathways derived from MetaCyc [34], [111] and experimental evidence from the Neurospora bibliome [37] were used to construct the first draft of the NeurosporaCyc Pathway/Genome database [112]. For the second stage, iterative phenotype-directed curation, we utilized FARM to suggest changes to the metabolic network based on a training set of experimentally observed growth phenotypes. These suggestions were reviewed manually, and accepted into the final model only if they were consistent with published experimental evidence. In the third stage, we independently validated the model based on a test set of experimentally observed viability phenotypes that were not utilized during model construction. In the fourth stage, we comprehensively predicted the phenotypes of all essential genes, nutrient rescues, and synthetic lethal interactions.
Figure 2.
(A) FBA does not require an input flux for cycles because it does not account for dilution of metabolites that participate in active reactions. (B) limed-FBA requires an input flux for cycles to compensate for dilution of metabolites that participate in active reactions. (C) FBA fails to correctly predict arg-14 gene essentiality because without an input flux, metabolite dilution prevents the isolated acetyl cycle compounds from being produced (side compounds not shown).
Figure 3.
Metabolic overview of Neurospora crassa.
The 257 metabolic pathways of Neurospora are divided into the 35 color-coded pathway classes. Biosynthetic pathways are displayed on the left, energy metabolism in the center, and degradation pathways are on the right. In addition to the cytosol and extracellular space, the model also contains 4 organelles: these are the vacuole, the nucleus, and the mitochondrion. The 299 transport reactions enable uptake and excretion of 137 metabolites and also exchange between the cytosol and each organelle.
Table 1.
Comparison of curation level among selected metabolic models.
Figure 4.
Minimal media gene essentiality predictions.
We curated a collection of mutant viability observations on minimal media and separated the collection into a training set, where knowledge of the viability phenotype was used to improve the model; and a test set, where the viability phenotype was hidden from the model. (A) Training and test set mutant viability observations were used to measure the sensitivity and specificity of the limed-FBA gene knockout viability predictions. While some inconsistencies were due to model error, several were resolved in the model's favor, as discussed in the text. (B) Using the same model, training and test sets, limed-FBA outperforms FBA and MD-FBA. (C) For comparison, we display the mutant viability prediction accuracies of previously published FBA models for S. cerevisiae and E. coli. Prediction accuracies of experimentally observed viability phenotypes that were used to optimize the model are displayed on the left panel [41], [42]. Prediction accuracies of viability phenotypes that were not explicitly used to construct the model are displayed on the right panel [62], [63].
Figure 5.
Prediction of nutrient rescue.
We curated a collection of conditions in which an auxotroph was rescued when minimal media was supplemented with a nutrient. We separated the collection into a training set, where knowledge of the rescue phenotype was used to improve the model, and a test set, where the rescue phenotype was hidden from the model. Because we only collected data on which nutrients rescued the auxotrophs, we could only measure sensitivity, not specificity. (A) Tables showing the sensitivity of limed-FBA predictions on nutrient rescue training and test sets. (B) Heatmap showing the growth phenotype of each mutant when minimal media is supplemented with each nutrient used in the training and test sets. Only mutants whose minimal media gene essentiality was correctly predicted are included. The minimal media used was Vogel's with sucrose as the carbon source except in the following cases: acu-3,5,6 genes are essential when acetate is the sole carbon source; oxD is essential when D-methionine is the sole sulfur source; nit-3 is essential when nitrate is the sole nitrogen source. Green squares indicate that the model's predictions were consistent with experiment; red squares indicate that the model failed to correctly predict growth; blue squares indicate potentially novel rescues; white squares indicate predictions of non-rescue. Striped squares show that the multi-substrate case does not contain additional information beyond the single-substrate case, e.g. methionine is predicted to rescue the cys-4 mutant, so methionine+threonine is also predicted to rescue cys-4.
Figure 6.
Mechanistic insight into the nutrient rescue of cysteine and methionine metabolism.
The model correctly predicts that cys-5, cys-9, and cys-11 mutants can be rescued when the downstream nutrients sulfite and thiosulfate are provided in the media. Similarly, the model correctly predicts that met-2, met-5, met-6, met7 and met-8 mutants are rescued by L-methionine; met-2, met-5 and met-7 mutants are rescued by L-homocysteine; and met-5 and met-7 mutants are rescued by L-cystathione. The model makes the potentially novel predictions that hom-1 and all cys mutants can be rescued by the downstream supplements L-cystathione, L-homocysteine and L-methionine. The model also makes the potentially novel prediction that cys-4 is not rescued by either upstream nutrient supplements sulfite or thiosulfate.
Figure 7.
Connection between glyoxylate cycle and gluconeogenesis reveals mechanistic insight into the nutrient rescue of acu mutants.
acu-3, acu-5 and acu-6 mutants are known to be lethal when acetate is the sole carbon source, because the glyoxylate cycle is blocked [88]. We correctly predict these mutants can be rescued by sucrose, and we additionally predict they can be rescued when supplemented with fructofuranose and glucose, because the enzymes encoded by acu-3, acu-5, and acu-6 are upstream of these sugars in the gluconeogenesis pathway.
Figure 8.
Supplementing with nutrients in alternate pathways can rescue some mutants.
The model makes the novel prediction that ace-2, ace-3, and ace-4 mutants (purple) in the TCA cycle can be rescued by supplementing minimal media with L-citrulline, L-arginine, L-ornithine, or L-glutamine (light blue) because each of these nutrients provide an alternate route via amino acid pathways to the essential metabolite 2-oxoglutarate (red).
Figure 9.
Synthetic lethality interaction map.
This gene-by-gene interaction map shows synthetic lethal predictions on Vogel's minimal media, except the double mutant pyr-1:uc-5 is on Vogel's+uracil. Shown are non-isozyme pairs, except the previously known isozyme pair cys-13:cys-14. If both synthetic lethal genes of a pair are in a common pathway, the square is cyan; if they are in interacting pathways, then it is colored orange. Validated synthetic lethal predictions have a black border.
Figure 10.
Mechanistic insight into three experimentally validated synthetic lethal auxotrophs and their nutrient rescue.
(A) The nitrogen assimilation pathway contains two alternate routes that convert α-ketoglutarate into the essential metabolite L-glutamine (red). (A1) The en(am)-2 mutant is viable, because α-ketoglutarate can be aminated to L-glutamate via am. (A2) The am mutant is viable, because α-ketoglutarate and L-glutamine can be converted to 2 L-glutamate via en(am)-2. (A3) The double mutant am:en(am)-2 is lethal when ammonium is the nitrogen source because both routes to L-glutamine are blocked, but (A4) can be rescued when the media is supplemented with L-glutamate (A4). (B) The only two routes for the synthesis of the essential metabolite L-proline are through arginine degradation and proline biosynthesis. (B1) The pro-3 mutant is blocked in proline biosynthesis, but can obtain L-proline through arginine degradation. (B2) The ota mutant is blocked in arginine degradation, but can obtain L-proline through proline biosynthesis. (B3) The double mutant pro-3:ota is blocked in both routes, but can be rescued when the nutrient media is supplemented with L-proline (B4). (C) There are only two biosynthetic routes to the essential metabolite uridine-5′-phosphate. (C1) The pyr-1 mutant can still obtain uridine-5′-phosphate from extracellular uracil, and the uc-5 mutant can obtain uridine-5′-phosphate from (S)-dihydroorotate (C2), but the pyr-1:uc-5 double mutant is blocked in both routes (C3). However, it can be rescued when the nutrient media is supplemented with uridine through its conversion to uridine-5′-phosphate in the pyrimidine salvage pathways (C4). Side compounds not shown.