Gap Detection for Genome-Scale Constraint-Based Models

BackgroundGenome-wide reconstructions of metabolism opened the way to thorough investigations of cell metabolism for health care and industrial purposes. However, the predictions offered by Flux Balance Analysis (FBA) can be strongly affected by the choice of flux boundaries, with particular regard to the flux of reactions that sink nutrients into the system. To mitigate possible errors introduced by a poor selection of such boundaries, a rational approach suggests to focus the modeling efforts on the pivotal ones.MethodsIn this work, we present a methodology for the automatic identification of the key fluxes in genome-wide constraint-based models, by means of variance-based sensitivity analysis. The goal is to identify the parameters for which a small perturbation entails a large variation of the model outcomes, also referred to as sensitive parameters. Due to the high number of FBA simulations that are necessary to assess sensitivity coefficients on genome-wide models, our method exploits a master-slave methodology that distributes the computation on massively multi-core architectures. We performed the following steps: (1) we determined the putative parameterizations of the genome-wide metabolic constraint-based model, using Saltelli’s method; (2) we applied FBA to each parameterized model, distributing the massive amount of calculations over multiple nodes by means of MPI; (3) we then recollected and exploited the results of all FBA runs to assess a global sensitivity analysis.ResultsWe show a proof-of-concept of our approach on latest genome-wide reconstructions of human metabolism Recon2.2 and Recon3D. We report that most sensitive parameters are mainly associated with the intake of essential amino acids in Recon2.2, whereas in Recon 3D they are associated largely with phospholipids. We also illustrate that in most cases there is a significant contribution of higher order effects.ConclusionOur results indicate that interaction effects between different model parameters exist, which should be taken into account especially at the stage of calibration of genome-wide models, supporting the importance of a global strategy of sensitivity analysis.

Determining intracellular metabolic flux through isotope labeling techniques such as 13C metabolic flux analysis (13C-MFA) incurs significant cost and effort. Previous studies have shown transcriptomic data coupled with constraint-based metabolic modeling can determine intracellular fluxes that correlate highly with 13C-MFA measured fluxes and can achieve higher accuracy than constraint-based metabolic modeling alone. These studies, however, used validation data limited to E. coli and S. cerevisiae grown on glucose, with significantly similar flux distribution for central metabolism. It is unclear whether those results apply to more diverse metabolisms, and therefore further, extensive validation is needed. In this paper, we formed a dataset of transcriptomic data coupled with corresponding 13C-MFA flux data for 21 experimental conditions in different unicellular organisms grown on varying carbon substrates and conditions. Three computational flux-balance analysis (FBA) methods were comparatively assessed. The results show when uptake rates of carbon sources and key metabolites are known, transcriptomic data provides no significant advantage over constraint-based metabolic modeling (average correlation coefficients, transcriptomic E-Flux2 0.725 and SPOT 0.650 vs non-transcriptomic pFBA 0.768). When uptake rates are unknown, however, predictions obtained utilizing transcriptomic data are generally good and significantly better than those obtained using constraint-based metabolic modeling alone (E-Flux2 0.385 and SPOT 0.583 vs pFBA 0.237). Thus, transcriptomic data coupled with constraint-based metabolic modeling is a promising method to obtain intracellular flux estimates in microorganisms, particularly in cases where uptake rates of key metabolites cannot be easily determined, such as for growth in complex media or in vivo conditions.

MotivationDetermining intracellular metabolic flux through isotope labeling techniques such as 13C metabolic flux analysis (13C-MFA) incurs significant cost and effort. Previous studies have shown transcriptomic data coupled with constraint-based metabolic modeling can determine intracellular fluxes that correlate highly with 13C-MFA measured fluxes and can achieve higher accuracy than constraint-based metabolic modeling alone. These studies, however, used validation data limited to E. coli and S. cerevisiae grown on glucose, with significantly similar flux distribution for central metabolism. It is unclear whether those results apply to more diverse metabolisms, and therefore further, extensive validation is needed.ResultsIn this paper, we formed a dataset of transcriptomic data coupled with corresponding 13C-MFA flux data for 21 experimental conditions in different unicellular organisms grown on varying carbon substrates and conditions. Three computational flux-balance analysis (FBA) methods were comparatively assessed. The results show when uptake rates of carbon sources and key metabolites are known, transcriptomic data provides no significant advantage over constraint-based metabolic modeling (average correlation coefficients, transcriptomic E-Flux2 0.725 and SPOT 0.650 vs non-transcriptomic pFBA 0.768). When uptake rates are unknown, however, predictions obtained utilizing transcriptomic data are generally good and significantly better than those obtained using constraint-based metabolic modeling alone (E-Flux2 0.385 and SPOT 0.583 vs pFBA 0.237). Thus, transcriptomic data coupled with constraint-based metabolic modeling is a promising method to obtain intracellular flux estimates in microorganisms, particularly in cases where uptake rates of key metabolites cannot be easily determined, such as for growth in complex media or in vivo conditions.

Constraint-based modeling is a well established modelling methodology used to analyze and study biological networks on both a medium and genome scale. Due to their large size, genome scale models are typically analysed using constraint-based optimization techniques. One widely used method is Flux Balance Analysis (FBA) which, for example, requires a modelling description to include: the definition of a stoichiometric matrix, an objective function and bounds on the values that fluxes can obtain at steady state. The Flux Balance Constraints (FBC) Package extends SBML Level 3 and provides a standardized format for the encoding, exchange and annotation of constraint-based models. It includes support for modelling concepts such as objective functions, flux bounds and model component annotation that facilitates reaction balancing. The FBC package establishes a base level for the unambiguous exchange of genome-scale, constraint-based models, that can be built upon by the community to meet future needs (e. g. by extending it to cover dynamic FBC models).

Understanding plant–microbe interactions is crucial for improving plants’ productivity and protection. Constraint-based metabolic modeling is one of the possible ways to investigate the bacterial adaptation to different ecological niches and may give insights into the metabolic versatility of plant pathogenic bacteria. We reconstructed a raw metabolic model of the emerging plant pathogenic bacterium Pectobacterium parmentieri SCC3193 with the use of KBase. The model was curated by using inParanoind and phenotypic data generated with the use of the OmniLog system. Metabolic modeling was performed through COBRApy Toolbox v. 0.10.1. The curated metabolic model of P. parmentieri SCC3193 is highly reliable, as in silico obtained results overlapped up to 91% with experimental data on carbon utilization phenotypes. By mean of flux balance analysis (FBA), we predicted the metabolic adaptation of P. parmentieri SCC3193 to two different ecological niches, relevant for the persistence and plant colonization by this bacterium: soil and the rhizosphere. We performed in silico gene deletions to predict the set of essential core genes for this bacterium to grow in such environments. We anticipate that our metabolic model will be a valuable element for defining a set of metabolic targets to control infection and spreading of this plant pathogen.

Metabolic network modeling is essential for understanding metabolic shifts occurring in complex physio-pathological processes. Currently, constraint-based modeling frameworks for metabolic networks primarily rely on Python or MATLAB libraries, requiring some coding skills. In contrast, more user-friendly tools lack essential features such as flux sampling or transcriptomic data integration. We introduce COBRAxy, a Python-based tool suite integrated into the Galaxy Project. COBRAxy enables constraint-based modeling and sampling techniques, allowing users to compute metabolic flux distributions for multiple biological samples. The tool also enables the integration of medium composition information to refine flux predictions. Additionally, COBRAxy provides a user-friendly interface for visualizing significant flux differences between populations on an enriched metabolic map. This extension provides a comprehensive and accessible framework for advanced metabolic analysis, enabling researchers without extensive programming expertise to explore complex metabolic processes. COBRAxy is available in the Galaxy ToolShed https://toolshed.g2.bx.psu.edu/view/bimib/cobraxy/9f78303dbd88.

Omic data analysis is steadily growing as a driver of basic and applied molecular biology research. Core to the interpretation of complex and heterogeneous biological phenotypes are computational approaches in the fields of statistics and machine learning. In parallel, constraint-based metabolic modeling has established itself as the main tool to investigate large-scale relationships between genotype, phenotype, and environment. The development and application of these methodological frameworks have occurred independently for the most part, whereas the potential of their integration for biological, biomedical, and biotechnological research is less known. Here, we describe how machine learning and constraint-based modeling can be combined, reviewing recent works at the intersection of both domains and discussing the mathematical and practical aspects involved. We overlap systematic classifications from both frameworks, making them accessible to nonexperts. Finally, we delineate potential future scenarios, propose new joint theoretical frameworks, and suggest concrete points of investigation for this joint subfield. A multiview approach merging experimental and knowledge-driven omic data through machine learning methods can incorporate key mechanistic information in an otherwise biologically-agnostic learning process.

The conversion of pentose to ethanol is one of the major barriers of industrializing lignocellulosic ethanol processes. As the most promising native strain for pentose fermentation, Scheffersomyces stipitis (formerly known as Pichia stipitis) has been widely studied for its xylose fermentation. In spite of the abundant experimental evidence regarding ethanol and by-products production under various aeration conditions, the mathematical descriptions of the processes are rare. In this work, the constraint-based metabolic network model for the central carbon metabolism of S. stipitis was reconstructed by integrating genomic (P. stipitis v2.0, KEGG), biochemical (ChEBI, KEGG) and physiological information available for this microorganisms and other related yeast. The model consists of the stoichiometry of metabolic reactions, the biosynthetic requirements for growth and other constraints. Flux Balance Analysis is applied to characterize the phenotypic behavior of S. stipitis grown on xylose. The model predictions are in good agreement with published experimental results. In addition, a series of specially designed in silico experiments are performed, and PCA has been applied to analyze the results to elucidate the redox balance of S. stipitis for xylose fermentation. The analysis revealed key metabolic reactions related to redox homeostasis and could provide important insights on cofactor engineering of xylose metabolism.

Summary Constraint-based modeling is a well established modelling methodology used to analyze and study biological networks on both a medium and genome scale. Due to their large size, genome scale models are typically analysed using constraint-based optimization techniques. One widely used method is Flux Balance Analysis (FBA) which, for example, requires a modelling description to include: the definition of a stoichiometric matrix, an objective function and bounds on the values that fluxes can obtain at steady state.The Flux Balance Constraints (FBC) Package extends SBML Level 3 and provides a standardized format for the encoding, exchange and annotation of constraint-based models. It includes support for modelling concepts such as objective functions, flux bounds and model component annotation that facilitates reaction balancing. The FBC package establishes a base level for the unambiguous exchange of genome-scale, constraint-based models, that can be built upon by the community to meet future needs (e. g. by extending it to cover dynamic FBC models).

Designing metabolic engineering strategies with genome-scale metabolic flux modeling Jiun Y Yen,1,2 Imen Tanniche,1 Amanda K Fisher,1–3 Glenda E Gillaspy,2 David R Bevan,2,3 Ryan S Senger,1 1Department of Biological Systems Engineering, 2Department of Biochemistry, 3Genomics, Bioinformatics, and Computational Biology Interdisciplinary Program, Virginia Tech, Blacksburg, VA, USA Abstract: New in silico tools that make use of genome-scale metabolic flux modeling are improving the design of metabolic engineering strategies. This review highlights the latest developments in this area, explains the interface between these in silico tools and the experimental implementation tools of metabolic engineers, and provides a way forward so that in silico predictions can better mimic reality and more experimental methods can be considered in simulation studies. The several methodologies for solving genome-scale models (eg, flux balance analysis [FBA], parsimonious FBA, flux variability analysis, and minimization of metabolic adjustment) all have unique advantages and applications. There are two basic approaches to designing metabolic engineering strategies in silico, and both have demonstrated success in the literature. The first involves: 1) making a genetic manipulation in a model; 2) testing for improved performance through simulation; and 3) iterating the process. The second approach has been used in more recently designed in silico tools and involves: 1) comparing metabolic flux profiles of a wild-type and ideally engineered state and 2) designing engineering strategies based on the differences in these flux profiles. Improvements in genome-scale modeling are anticipated in areas such as the inclusion of all relevant cellular machinery, the ability to understand and anticipate the results of combinatorial enrichment experiments, and constructing dynamic and flexible biomass equations that can respond to environmental and genetic manipulations. Keywords: genome-scale modeling, genome-scale modeling, flux balance analysis, flux variability analysis, minimization of metabolic adjustment, metabolic bottleneck, pathway optimization

Flux balance analysis (FBA) has been used extensively to analyze genome-scale, constraint-based models of metabolism in a variety of organisms. The predictive accuracy of such models has recently been improved through the integration of high-throughput expression profiles of metabolic genes and proteins. However, extensions of FBA often require that such data be discretized a priori into sets of genes or proteins that are either 'on' or 'off'. This procedure requires selecting relatively subjective expression thresholds, often requiring several iterations and refinements to capture the expression dynamics and retain model functionality. We present a method for mapping expression data from a set of environmental, genetic or temporal conditions onto a metabolic network model without the need for arbitrary expression thresholds. Metabolic Adjustment by Differential Expression (MADE) uses the statistical significance of changes in gene or protein expression to create a functional metabolic model that most accurately recapitulates the expression dynamics. MADE was used to generate a series of models that reflect the metabolic adjustments seen in the transition from fermentative- to glycerol-based respiration in Saccharomyces cerevisiae. The calculated gene states match 98.7% of possible changes in expression, and the resulting models capture functional characteristics of the metabolic shift. MADE is implemented in Matlab and requires a mixed-integer linear program solver. Source code is freely available at http://www.bme.virginia.edu/csbl/downloads/.

Constraint-based reconstruction and analysis (COBRA) is a powerful systems biology approach for computational bioengineering. Synechococcus elongatus PCC 11801 and PCC 11802 are fast-growing, stress-tolerant cyanobacteria that are promising platforms for photosynthetic biomanufacturing. Here, we present constraint-based models (CBMs) iLV1052 and iLV1087 of PCC 11801 and PCC 11802, respectively, to facilitate and streamline strain engineering efforts. Following draft reconstruction using a template model, the models underwent extensive manual curation to reduce redundancy, and verification using BiGG, KEGG, and BRENDA databases. We added 281 and 69 new reactions for PCC 11801 and PCC 11802, respectively, associated with stress tolerance, growth stability, antioxidant defense, energy regulation, and sulfur acquisition. The models were refined through iterative debugging and validation using flux balance analysis, flux variability analysis, and single gene/reaction deletion analysis. Gene essentiality predictions gave 69% accuracy for PCC 11801 and 83% for PCC 11802. The flux maps captured key features of cyanobacterial metabolism, including an incomplete TCA cycle. The final PCC 11801 CBM contained 1130 reactions, 1052 genes, and 930 metabolites, while the PCC 11802 CBM included 1199 reactions, 1087 genes, and 951 metabolites. The simulations predicted that succinic acid exhibited the highest theoretical yield among the tested target products in both strains. Using the Optknock framework, phosphoenolpyruvate carboxylase was identified as a metabolic hotspot for future bioengineering efforts aimed at the production of valuable products like ethanol, butanol, and butanediol.

Regulatory dynamic enzyme-cost flux balance analysis: A unifying framework for constraint-based modeling

Constraint-based models of metabolism have been used in a variety of studies on drug discovery, metabolic engineering, evolution, and multi-species interactions. These genome-scale models can be generated for any sequenced organism since their main parameters (i.e., reaction stoichiometry) are highly conserved. Their relatively low parameter requirement makes these models easy to develop; however, these models often result in a solution space with multiple possible flux distributions, making it difficult to determine the precise flux state in the cell. Recent research efforts in this modeling field have investigated how additional experimental data, including gene expression, protein expression, metabolite concentrations, and kinetic parameters, can be used to reduce the solution space. This mini-review provides a summary of the data-driven computational approaches that are available for reducing the solution space and thereby improve predictions of intracellular fluxes by constraint-based models.

For nearly all of the 20th century, biologists gained considerable insights into the fundamental principles of cellular dynamics by examining select modules of biochemical processes. This form of analysis provides detailed information about the workings of the examined pathways. However, any attempt to alter the normal function of bacteria (perhaps for industrial or medicinal goals) requires a detailed global understanding of cellular mechanisms. The reductionist mode of analysis cannot provide the required information for developing the needed perspective on the complex interactions of biochemical pathways. Thankfully, the increasing availability of microbial genomic, transcriptomic, proteomic and other high-throughput data permits system-level analyses of microbiology. During the past two decades, systems biologists have developed constraint-based genome-scale models (GSM) of metabolism for a variety of pathogens. These models are important tools for assessing the metabolic capabilities of various genotypes. Simultaneously, new computational methods have been developed that use these network reconstructions to answer an array of important immunological questions. The objective of this article is to briefly review some of the uses of GSMs for studying bacterial metabolism under different conditions and to discuss how the calculated solutions can be used for rational design of drugs.