BackgroundGenome-wide sensitivity screens in yeast have been immensely popular following the construction of a collection of deletion mutants of non-essential genes. However, the auxotrophic markers in this collection preclude experiments on minimal growth medium, one of the most informative metabolic environments. Here we present quantitative growth analysis for mutants in all 4,772 non-essential genes from our prototrophic deletion collection across a large set of metabolic conditions.ResultsThe complete collection was grown in environments consisting of one of four possible carbon sources paired with one of seven nitrogen sources, for a total of 28 different well-defined metabolic environments. The relative contributions to mutants' fitness of each carbon and nitrogen source were determined using multivariate statistical methods. The mutant profiling recovered known and novel genes specific to the processing of nutrients and accurately predicted functional relationships, especially for metabolic functions. A benchmark of genome-scale metabolic network modeling is also given to demonstrate the level of agreement between current in silico predictions and hitherto unavailable experimental data.ConclusionsThese data address a fundamental deficiency in our understanding of the model eukaryote Saccharomyces cerevisiae and its response to the most basic of environments. While choice of carbon source has the greatest impact on cell growth, specific effects due to nitrogen source and interactions between the nutrients are frequent. We demonstrate utility in characterizing genes of unknown function and illustrate how these data can be integrated with other whole-genome screens to interpret similarities between seemingly diverse perturbation types.

The green picoalga Ostreococcus is emerging as a simple plant model organism, and two species, O. lucimarinus and O. tauri, have now been sequenced and annotated manually. To evaluate the completeness of the metabolic annotation of both species, metabolic networks of O. lucimarinus and O. tauri were reconstructed from the KEGG database, thermodynamically constrained, elementally balanced, and functionally evaluated. The draft networks contained extensive gaps and, in the case of O. tauri, no biomass components could be produced due to an incomplete Calvin cycle. To find and remove gaps from the networks, an extensive reference biochemical reaction database was assembled using a stepwise approach that minimized the inclusion of microbial reactions. Gaps were then removed from both Ostreococcus networks using two existing gap-filling methodologies. In the first method, a bottom-up approach, a minimal list of reactions was added to each model to enable the production of all metabolites included in our biomass equation. In the second method, a top-down approach, all reactions in the reference database were added to the target networks and subsequently trimmed away based on the sequence alignment scores of identified orthologues. Because current gap-filling methods do not produce unique solutions, a quality metric that includes a weighting for phylogenetic distance and sequence similarity was developed to distinguish between gap-filling results automatically. The draft O. lucimarinus and O. tauri networks required the addition of 56 and 70 reactions, respectively, in order to produce the same biomass precursor metabolites that were produced by our plant reference database.

Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA). TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1) previous genome-wide direct gene-essentiality assignments; and, 2) flux balance analysis (FBA) predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.

Background: Genome-scale draft metabolic networks are incomplete, even for well studied organisms. Results: Reactions selected by minimizing flux through unlikely reactions resulted in networks of superior quality. Conclusion: Genome-scale models have many network completion solutions but require the addition of unsupported reactions to be functional. Significance: Metabolic networks guide synthetic biology efforts, and the quality of networks determines their predictive power.

In pursuit of establishing a realistic metabolic phenotypic space, the reversibility of reactions is thermodynamically constrained in modern metabolic networks. The reversibility constraints follow from heuristic thermodynamic poise approximations that take anticipated cellular metabolite concentration ranges into account. Because constraints reduce the feasible space, draft metabolic network reconstructions may need more extensive reconciliation, and a larger number of genes may become essential. Notwithstanding ubiquitous application, the effect of reversibility constraints on the predictive capabilities of metabolic networks has not been investigated in detail. Instead, work has focused on the implementation and validation of the thermodynamic poise calculation itself. With the advance of fast linear programming-based network reconciliation, the effects of reversibility constraints on network reconciliation and gene essentiality predictions have become feasible and are the subject of this study. Networks with thermodynamically informed reversibility constraints outperformed gene essentiality predictions compared to networks that were constrained with randomly shuffled constraints. Unconstrained networks predicted gene essentiality as accurately as thermodynamically constrained networks, but predicted substantially fewer essential genes. Networks that were reconciled with sequence similarity data and strongly enforced reversibility constraints outperformed all other networks. We conclude that metabolic network analysis confirmed the validity of the thermodynamic constraints, and that thermodynamic poise information is actionable during network reconciliation.