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The RAVEN Toolbox and Its Use for Generating a Genome-scale Metabolic Model for Penicillium chrysogenum

The RAVEN Toolbox and Its Use for Generating a Genome-scale Metabolic Model for Penicillium... We present the RAVEN (Reconstruction, Analysis and Visualization of Metabolic Networks) Toolbox: a software suite that allows for semi-automated reconstruction of genome-scale models. It makes use of published models and/or the KEGG database, coupled with extensive gap-filling and quality control features. The software suite also contains methods for visualizing simulation results and omics data, as well as a range of methods for performing simulations and analyzing the results. The software is a useful tool for system-wide data analysis in a metabolic context and for streamlined reconstruction of metabolic networks based on protein homology. The RAVEN Toolbox workflow was applied in order to reconstruct a genome-scale metabolic model for the important microbial cell factory Penicillium chrysogenum Wisconsin54-1255. The model was validated in a bibliomic study of in total 440 references, and it comprises 1471 unique biochemical reactions and 1006 ORFs. It was then used to study the roles of ATP and NADPH in the biosynthesis of penicillin, and to identify potential metabolic engineering targets for maximization of penicillin production. Citation: Agren R, Liu L, Shoaie S, Vongsangnak W, Nookaew I, et al. (2013) The RAVEN Toolbox and Its Use for Generating a Genome-scale Metabolic Model for Penicillium chrysogenum. PLoS Comput Biol 9(3): e1002980. doi:10.1371/journal.pcbi.1002980 Editor: Costas D. Maranas, The Pennsylvania State University, United States of America Received September 11, 2012; Accepted January 24, 2013; Published March 21, 2013 Copyright:  2013 Agren et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project has been financed by European Research Council (Grant 247013), the EU-funded project SYSINBIO and Sandoz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] automated approaches to model reconstruction. There are now a Introduction number of tools available for automated annotation of genes Genome sequencing projects have in recent years contributed [13,14]. However, the annotated genes must be linked to enormously to our understanding of the metabolic capabilities of metabolic reactions in a way so as to generate a functional cellular systems. Functional annotation of the gene products allow metabolic model. This includes the addition of spontaneous for reconstruction of genome-scale metabolic models (GEMs) that reactions and non-carrier mediated transport across membranes as summarize these metabolic capabilities in a consistent and well as sub-cellular localization of enzymes. Most importantly, the compact way [1,2]. A number of mathematical tools, including model must also be constructed in a way so that all reactions are sampling of available metabolic states [3,4] and methods borrowed balanced and well-connected [15]. This tends to become a from computational geometry [5], have been developed to analyze problem if the gene-reaction relationship is automatically inferred the resulting networks and to gain insight into the complex from databases, partly due to differences in metabolite naming, interactions that give rise to the metabolic capabilities. GEMs have but mainly because of how complex carbohydrates and complex also been used extensively for simulation of metabolism, partic- lipids are represented. Because of the aforementioned issues it ularly for metabolic engineering purposes [6,7]. Since these makes sense to use previously reconstructed models as templates models connect metabolites, proteins, and genes they are for new GEMs. Here we present the RAVEN Toolbox, which particularly well suited for the integration of metabolomics, allows the user to input GEM(s) for one or more template proteomics, and genomics which is, in a sense, the goal of systems organisms, their corresponding protein sequences, and the protein biology [8,9]. sequences of the target organism. A GEM for the target organism The foundation of a GEM is the functional annotation of the is then constructed based on orthology between the protein genes. The first GEMs were primarily for model organisms for sequences of the target organism and the organisms of the which direct evidence exists in the literature for a large proportion template models. Metabolic functions not present in the template of the genomically encoded functions [10,11]. However, as the models can obviously not appear in the new model, and to account number of genome sequencing projects increases there is a for these missing reactions the RAVEN Toolbox also includes a growing demand of GEMs for less well known organisms. These functionality that matches proteins to KEGG Orthology (KO) models must by necessity be built largely relying on protein categories [16] by using Hidden Markov models to capture the homology to more well-characterized organisms [12]. This, representative amino acid pattern in each KO. The resulting together with the large amount of manual work that is involved metabolic network can be used for automatic or manual gap in a strict bottom-up reconstruction, has sparked interest in more filling, or it can be used on its own as a draft network. PLOS Computational Biology | www.ploscompbiol.org 1 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox for the reconstruction of a GEM of the industrially important mold Author Summary Penicillium chrysogenum. The Penicillium genus encompasses species of Genome-scale models (GEMs) are large stoichiometric great economical, medical, and environmental importance [22]. models of cell metabolism, where the goal is to incorpo- Members of the Penicillium genus serve important roles in the food rate every metabolic transformation that an organism can industry, both as some of the main spoilers of fresh vegetables and perform. Such models have been extensively used for the as essential actors in the production of blue cheeses. Most study of bacterial metabolism, in particular for metabolic importantly though, they are sources of major antibiotics, engineering purposes. More recently, the use of GEMs for particularly penicillin and griseofulvin. eukaryotic organisms has become increasingly wide- The industrial production of b-lactam antibiotics, such as spread. Since these models typically involve thousands of penicillins and cephalosporins, is one of the success stories of metabolic reactions, the reconstruction and validation of biotechnology. Today the b-lactams represent one of the largest them can be a very complex task. We have developed a biotechnological products in terms of value, with sales of about software suite, RAVEN Toolbox, which aims at automating USD 15 billion [23]. The industrial P. chrysogenum strains have parts of the reconstruction process in order to allow for been subjected to 50 years of directed evolution to increase the faster reconstruction of high-quality GEMs. The software is yields and titers of penicillin, with great cost reduction and particularly well suited for reconstruction of models for productivity gain, but the yields are still far from the theoretical eukaryotic organisms, due to how it deals with sub-cellular maximum [24]. A GEM of P. chrysogenum could aid in identification localization of reactions. We used the software for reconstructing a model of the filamentous fungi Penicillium of metabolic bottlenecks as well as in elucidating the underlying chrysogenum, the organism used in penicillin production reason for the significantly better performance of industrial strains and an important microbial cell factory. The resulting compared to low producing strains. model was validated through an extensive literature survey and by comparison with published fermentation Results data. The model was used for the identification of transcriptionally regulated metabolic bottlenecks in order The RAVEN Toolbox to increase the yield in penicillin fermentations. In this A software suite named the RAVEN Toolbox (Reconstruction, paper we present the RAVEN Toolbox and the GEM for P. Analysis, and Visualization of Metabolic Networks) was developed. chrysogenum. The toolbox is a complete environment for reconstruction, analysis, simulation, and visualization of GEMs and runs within MATLAB. The software imports and exports models in two Several approaches which also aim at generating GEMs from either a template model or from a general database have been formats: the widely used Systems Biology Markup Language published [17–20]. Table 1 summarizes the capabilities of the (SBML) format [25] and a Microsoft Excel model representation. RAVEN Toolbox compared to some other published approaches Both these formats allow for extensive annotation of model when it comes to automatic reconstruction. However, the largest components, such as International Chemical Identifier strings difference is maybe not in the approaches taken, but in that the (InChI) [26] for metabolites or database identifiers for reactions RAVEN Toolbox is a complete software for all tasks involving and genes. The native model format for the RAVEN Toolbox reconstruction and simulation of GEMs. In this aspect the RAVEN follows the format of the yeast consensus metabolic network [27], Toolbox is more similar to the COBRA Toolbox, but with extensive but models in the COBRA Toolbox format can also be imported reconstruction capabilities [21]. Even though the RAVEN Toolbox [21]. The Microsoft Excel representation enables the user to set can be used for fully automated reconstruction, in a manner similar simulation parameters such as bounds and objective function to Model SEED, the intended purpose is to make use of the coefficients directly in the spread sheets. This simplifies the extensive quality control and gap identification/gap filling features modeling process for users not comfortable with working within a for increasing the quality of reconstructions, as well as for decreasing scripting environment, as well as providing a simpler, but less the time needed for reconstructing high-quality models. rigorous, model format compared to SBML. The software, The RAVEN Toolbox was evaluated for its ability to together with a manual, a set of tutorials and a detailed description reconstruct a GEM for the well studied yeast Saccharomyces cerevisiae, of the supported file formats is available through the BioMet and the resulting GEM was compared with a manually Toolbox [28] (http://www.sysbio.se/BioMet). Figure 1 summa- reconstructed model. Thereafter the RAVEN toolbox was used rizes the capabilities of the RAVEN Toolbox. Table 1. Comparison between the RAVEN Toolbox and other software for automatic GEM reconstruction. RAVEN Model SEED [20] AUTOGRAPH [18] IdentiCS [60] GEM System [17] Includes general network X X X X Generates functional models X X Assigns sub-cellular localization X Can use user defined models X X Integrates gap filling X X X Offline software X X Includes visualization X X X Gene prediction XX doi:10.1371/journal.pcbi.1002980.t001 PLOS Computational Biology | www.ploscompbiol.org 2 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox Figure 1. The RAVEN Toolbox. The software allows for reconstruction of GEMs based on template models or on the KEGG database. The resulting models can be exported to a number of formats, or they can be used for various types of simulations. The RAVEN Toolbox has a strong focus on quality control. Visualization of simulation result and/or integration of other types of data can be performed by overlaying information on pre-drawn metabolic maps. The software also implements the INIT algorithm, which is a powerful approach for reconstruction of tissue-specific models [59]. HMM: Hidden Markov model, LP: Linear programming, QP: Quadratic programming, MILP: Mixed-integer linear programming. doi:10.1371/journal.pcbi.1002980.g001 The software has three main foci: 1) automatic reconstruction of GEMs are also typically constructed for modeling purposes, which is GEMs based on protein homology, 2) network analysis, modeling not the case for reaction databases. The downside is that only and interpretation of simulation results, 3) visualization of GEMs reactions present in the template models can be included. The using pre-drawn metabolic network maps. RAVEN Toolbox therefore contains two approaches for automatic Automated reconstruction of GEMs based on protein generation of draft models; while the method mentioned above relies homology. Previously published GEMs represent a solid basis on the metabolic functions represented in previously published for metabolic reconstruction of models for new organisms, in models, the complementary method uses the KEGG database for particular if the organisms are closely related and therefore share automatic identification of new metabolic functions that are not many metabolic capabilities. The main advantage of using existing included in the published models. models compared to reaction databases, such as KEGG or The first approach lets the user supply a number of existing BRENDA [29], is that they contain information that can be GEMs and FASTA files with protein sequences for the template difficult to obtain in an automated manner, in particular models and for the organism of interest. The software then directionality and compartmentalization. There have been generates a draft model based on protein orthology. The default attempts to predict the directionality of reactions based on the implementation uses bi-directional BLASTp [31] for evaluation of estimates of the standard Gibbs energies of formation for the protein homology, but the software also supports other homology involved metabolites [30]. However, we believe that manually measurements as long as a score can be assigned to each pair wise reconstructed networks for related species can be a more reliable protein comparison. The resulting model can be exported as a source of directionality information. The same is true for SBML file or be used in MATLAB for simulation and further analysis. compartmentalization. Even though the RAVEN Toolbox con- tains methods for inferring subcellular localization based on The second approach is also based on protein homology but predictors, it is to be viewed as an aid rather than an exact method. requires no template models. Instead it relies on the information PLOS Computational Biology | www.ploscompbiol.org 3 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox on protein sequences and on the assigned metabolic reactions that of any metabolite. The solutions can then be cross-referenced is available in the KEGG database. The method makes use of the to balancing information from getElementalBalance in order to KEGG Orthology (KO) IDs, which are manually annotated sets of identify reactions which are both active and have wrong/ genes that encode some specified metabolic function. Each KO is lacking composition. This process can also be done automat- associated with a number of metabolic reactions. The aim of the ically using removeBadRxns. present method is to assign genes to these KOs based on the After the user has added relevant exchange reactions consensus protein sequence. The tool first downloads all relevant canProduce/canConsume can be used to generate a list of the parts of the KEGG database to a local directory and parses these metabolites that can have net synthesis or consumption. Early files to generate a GEM representing a metabolic network across on in the reconstruction process it is likely that not all biomass all of the species annotated in KEGG, i.e. this would lead to a precursors can be synthesized. The function checkProduction can network comprising 7029 metabolites, 8398 reactions and 843369 be very useful in this situation. It calculates the smallest set of genes, when using the most current version of the KEGG metabolites which must have net synthesis in order to enable database. A GEM for the organism of interest is then constructed net synthesis of all other metabolites. This gives the user by choosing a subset of this larger model and linking the reactions information such as ‘‘in order to synthesize biomass, you must with the corresponding genes. The protein sequences for each KO enable synthesis of valine and coenzyme A’’ or ‘‘if synthesis of are retrieved and aligned using MUSCLE [32]. The user has the choline is enabled, the following set of metabolites could also option to only use genes from organisms of a given phylogenetic be synthesized’’. The function also allows the user to set rules distance from the target organism, e.g. only fungal genes or only about merging compartments, since it can be easier to first eukaryotic genes. A hidden Markov model is then generated based make sure that the model is functional with merged on the sequences for each KO using HMMER [33]. The final step compartments and deal with transport and sub-cellular is the querying of the set of HMMs with the protein sequences of localization afterwards. the organism of interest. If a gene has a significant match to one Ideally all reactions should be able to carry flux if all relevant KO, the reactions associated to that KO are added to the model exchange reactions are available. The function haveFlux can be together with the corresponding gene. This process is fully used to identify reactions which cannot carry flux, and also to automated, and the user only needs to supply a FASTA file with distinguish between reactions which cannot carry flux because protein sequences. Users who do not subscribe to KEGG can some substrate cannot be synthesized and those which cannot download pre-trained HMMs for eukaryotes and prokaryotes carry flux because some product cannot be further consumed. through the BioMet Toolbox (http://www.sysbio.se/BioMet). However, because of the many internal loops in GEMs it is These HMMs are based on the last open version of KEGG. common that reactions can carry flux and appear well- More advanced users can set parameters that affect how genes are connected even if they are not connected to the rest of the mapped to KOs and how general, unbalanced, or otherwise metabolic network. getAllSubGraphs can be used to identify such problematic reactions from KEGG should be dealt with. subnetworks using Tarjan’s algorithm [34]. Model analysis and simulation. The approach proposed The function fillGaps can be used to retrieve reactions from a above will facilitate and accelerate the generation of a draft set of template models or from KEGG in order to generate a metabolic network reconstruction. The automated reconstruction functional network. The user can set constraints on their can lead to some loss of control compared to a stricter manual, model, such as that it should be able to produce biomass from bottom-up approach. It is therefore important to identify and fill minimal media, and fillGaps will then solve the MILP problem gaps in the model to ensure that the network is functioning as of including the minimal set of reactions from a set of template required. In a high quality model all reactions should be able to models in order to satisfy the constraints. The same function have a flux if all uptake and excretion reactions are allowed and can be used to enable net synthesis of all metabolites or to net synthesis of most metabolites should be possible (the exception enable flux through all reactions. This approach is similar to would normally be some co-factors). The second criterion is that taken in Model SEED, and enables fully automatic model important, since the large degree of freedom in GEMs allow for reconstruction. However, we suggest that GEM reconstruction internal loops where reactions can carry flux but where no net should be done iteratively and with manual input and that the consumption or synthesis of metabolites occurs. The RAVEN results from these algorithms are to be viewed as suggestions to Toolbox contains a number of methods to support the gap filling point the user in the right direction. process. The following section describes the suggested workflow for In eukaryotes the enzymatic reactions are distributed between gap identification and filling when starting from a draft network. N different organelles. To determine which reactions occur Gap filling traditionally centers on adding reactions in order to where is a difficult task, and one of the more time-consuming enable production of all precursors needed for biomass steps in the reconstruction process. The RAVEN Toolbox production. However, it is equally important to ensure that takes a first step towards speeding up this step by including a the model cannot produce anything when there is no uptake of method for assigning subcellular localization to enzymatic metabolites. The reactions which enable this type of behavior reactions in an automated fashion. The algorithm aims at are typically those which involve polymers, metabolite pools, assigning localization in a manner that is consistent with signal or other abstract metabolites but they can also simply be peptide composition and physiochemical protein properties, erroneous reactions. A brute force solution would be to while at the same time maintaining a well-connected and exclude all reactions which are not elementally balanced, but functional network. The default predictor is WoLF PSORT, this could result in a large fraction of the network being which is distributed with the RAVEN Toolbox [35]. A parser deleted, as many metabolites typically lack information about for other predictors, such as CELLO is also included [36]. In elemental composition. The makeSomething and consumeSomething short, the algorithm works by generating fully connected functions identifies such reactions by solving the mixed integer solutions, which are then scored based on the agreement to the linear programming (MILP) problem of finding the smallest set predicted localization and the number of transport reactions of reactions which results in the net synthesis or consumption which had to be included in order to have a connected PLOS Computational Biology | www.ploscompbiol.org 4 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox network. The problem is solved using simulated annealing. A Validation of the workflow. The RAVEN Toolbox pipeline was validated by constructing a model for Saccharomyces cerevisiae,a more detailed description is available in Figure 2. model organism for which several GEMs have been constructed. The RAVEN Toolbox also contains a number of methods for To compare the quality of the automatically generated model to a performing simulations using GEMs. In this aspect it is similar to manually curated one, some kind of reference was needed. As all the COBRA Toolbox [21]. Most of the features of COBRA models contain errors it would not be very relevant to simply Toolbox are also present in the RAVEN Toolbox, with the compare the similarity between the RAVEN Toolbox generated exception of dynamic FBA. This includes linear programming model and a previously published model. Saccharomyces Genome such as FBA, quadratic programming such as MoMA, mixed Database (SGD) was therefore used as a reference with respect to integer linear programming applications, and random sampling. the enzymes present in S. cerevisiae and their subcellular localiza- Utility functions such as setting constraints and objectives, adding tion. A model was generated from KEGG in a fully automatic or removing model elements, presenting simulation outputs, manner and then compared to the iIN800 model, a model which sensitivity analysis, screening for gene deletions, and fitting model has been shown to have excellent simulation capabilities [37]. It parameters such as maintenance ATP consumption are also should be noted that this fully automatic reconstruction is not how included. For a full description of all functions of the toolbox, see the RAVEN Toolbox is intended to be used for reconstruction. the supplied manual. The RAVEN Toolbox uses MOSEK We suggest that the user view the output of each step as (MOSEK ApS, Copenhagen, Denmark) for solving the underlying suggestions, and manually fill gaps or fix problematic reactions. optimization problems. MOSEK is proprietary software but a full However, we wanted to perform an evaluation of the overall featured license is freely available for academic use. reconstruction feature of the RAVEN Toolbox. Figure 2. Prediction of subcellular localization of reactions. Circles correspond to metabolites and lines correspond to reactions. Green lines are reactions which are in their correct compartment according to the predictions. Red are reactions which are in an incorrect compartment and orange are reactions where there is no strong indication for either compartment. A) There is a tradeoff between connectivity and agreement with predicted localization. Network 1 represents the extreme case where connectivity is much more important than predicted localization scores. All reactions are then localized to the cytosol. Network 2 represents the other extreme case where the reactions are localized only based on localization scores and with no regard for connectivity. This would result in an unconnected network. Network 3 represents the case where the network is connected, while still being in good agreement with the localization scores. The underlying assumption in the algorithm is that a good network is characterized by being fully connected, in the sense that all metabolites are synthesized in at least one reaction and consumed in at least one reaction, while still being in good agreement with the localization scores and relying on the smallest possible number of transport reactions to achieve this. B) Summary of the localization algorithm. 1. The algorithm first randomly moves one gene product and its associated reaction(s) to another compartment. The probabilities depend on the scores for the gene products in their respective compartments. 2. This may result in an unconnected network. The algorithm then tries to find a small set of reactions which, when moved, reconnects the network. If moving these reactions would result in a large decrease of fitness, then the network is connected by including transport reactions for some metabolites instead. 3. The connected network is then scored as the sum of scores for all genes in their assigned compartment, minus the cost of all transport reactions that had to be included in order to keep the network connected. The user can set the relative weight given to transport compared to gene localization. The overall problem is solved using simulated annealing. doi:10.1371/journal.pcbi.1002980.g002 PLOS Computational Biology | www.ploscompbiol.org 5 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox The model was generated using getKEGGModelForOrganism with The RAVEN Toolbox also contains a method for partitioning the settings to only use eukaryotic genes when training the HMMs, enzymatic reactions to compartments in a manner that keeps the a cutoff of 1e-30 when matching genes to the HMMs, and to network connected, but at the same time in agreement with the exclude reactions labeled as general or incomplete in KEGG. S. results from predictors of protein localization (see Figure 2 for cerevisiae genes were excluded in the training of the HMMs to details). The default predictor, WoLF PSORT, was used to predict simulate reconstruction of an organism for which there is little the protein localization of all ORFs in the FASTA file. previous gene annotation. Not all unbalanced or erroneous predictLocalization was then used to partition the network between reactions were labeled as such, and this resulted in that the mitochondria and cytosol. The transport cost was set to 0.1. Table KEGG model could produce some metabolites without any S6 lists the genes for which the corresponding reactions were uptakes. removeBadRxns identified 79 reactions which enabled such assigned to the mitochondria. 119 gene products were assigned to production (see Table S1). Out of these 72 were unbalanced, the mitochondria and the remaining 594 gene products were general or polymer reactions and as such were correctly removed. assigned to the cytosol. Out of the 119 predicted mitochondrial 7 reactions were correct, but lacked composition about the gene products, 72% were listed as mitochondrial in the SGD metabolites (it is a setting in removeBadRxns whether it is allowed to based on experimental evidence. The same calculations for iIN800 remove such reactions). give that 91 gene products are mitochondrial and that 83% were Based on experimental minimal media the model was allowed listed as mitochondrial in SGD. Localization predictions based uptake of glucose, phosphate, sulfate, NH3, oxygen and the only on primary protein sequences are not very exact, and the essential nutrients 4-aminobenzoate, riboflavin, thiamine, biotin, resulting model from predictLocalization will not be totally biolog- folate, and nicotinate [38]. Uptake of the carriers carnitine and ically correct. The main issue is that all transport reactions are acyl-carrier protein was allowed for modeling purposes (many formulated as passive diffusion, while in reality other types of compounds are bound to them and therefore net synthesis of these transport are also taking place. However, the method is able to compounds is not possible without them). This was the only quickly generate a connected model where the enzyme localiza- manual step in the reconstruction of the yeast model. tions are in almost as good agreement to SGD as a published model. This could be useful for many applications, such as when The resulting model contained 1126 reactions, 1144 metabolites and 713 genes (before compartmentalization). 521 (73%) of those using metabolic networks for integrating omics data, and it constitutes a first step towards fully automated reconstruction of genes were shared with iIN800. 192 genes were unique to the automatically reconstructed model and 91 genes were unique to eukaryote GEMs. the iIN800 model (since there are no transport reactions in These results show both strengths and weaknesses of using a KEGG, all transporters were excluded from iIN800 for the fully automatic approach to reconstruction. A model capable of purpose of this comparison). Figure 3 shows a classification of the producing all the needed building blocks for synthesis of protein, genes that are unique to either the automatically reconstructed RNA, DNA, and the cell wall was generated solely from a FASTA model or to iIN800 (see Table S2 for details). As can be seen, the file and with almost no manual input. The automated gap filling automatically reconstructed model has a significantly larger identified 17 new reactions, out of which 8 were not present in the proportion of enzymes compared to the published model. published S. cerevisiae model. As was shown in Figure 3, the quality in terms of included genes was as good or better compared to the Given the inputs the model could have net-synthesis of 476 (42%) of the 1144 metabolites (see Table S3 for details). As a published model. On the other hand, the gap filling included 19 comparison, 456 (66%) out of 683 unique metabolites in iIN800 reactions which did not belong in the model, and complex lipids could be synthesized given thesameinputs. Amongthe 476 could not be synthesized. The sub-cellular localization of enzymes metabolites were 19 out of the 22 standard amino acids (leucine, was up to par with the published model, but with the drawback methionine, and taurine could not be synthesized), the that all transport reactions were formulated as passive diffusion. In nucleotides needed for RNA and DNA synthesis (ATP, GTP, a real situation a reconstruction should therefore be done in an CTP, UTP, dATP, dGTP, dCTP, and dTTP), fatty acids, iterative manner, with manual input after each iteration (the user sterols such as lanosterol and ergosterol, important co-factors would, for example, remove the 19 bad reactions from the such as NADH, NADPH, FADH2 and CoA, and the building template model and then run fillGaps again). blocks needed forcellwallassembly (UDP-glucose, UDP-N- Visualization of GEMs. Stoichiometric metabolic models acetyl-D-glucosamine and mannose). The only major biomass have been proven to generate remarkably good predictions when constituents that could not be synthesized were complex lipid it comes to the central carbon metabolism in microorganisms. compounds such as phospholipids and sphingolipids. This is However, the lack of kinetic and regulatory information is a rather because of the combinatorial nature of fatty acid metabolism large simplification and it is possible to get simulation results that (given ,20 fatty acids there are 20!/2!(20-2) = 190 possible have little biological meaning (such as thermodynamically versions of phosphatidylcholine) and how it is represented in disallowed loops). It is therefore imperative to understand the KEGG. underlying reasons for a change in predicted phenotype after a As the next step of the fully automatic reconstruction, fillGaps perturbation such as a gene deletion. Due to the large was used to automatically fill gaps in the yeast network using the dimensionality of GEMs interpretation of flux distributions is a full KEGG database as a template. This resulted in 45 reactions rather daunting task. Visualization of fluxes can aid with being added, which in turn enabled the synthesis of 91 metabolites interpretation, as well as provide an instant overview of how the that could previously not be synthesized (see Table S4). Among system functions. Software that aims at network visualization them were the three amino acids that were previously missing. A based purely on connectivity, such as Cytoscape [39] or closer investigation of the reactions which were added (see Table CellDesigner [40], cannot provide a comprehensible or well S5) showed that out of the 45 added reactions, 17 had evidence to organized image of GEMs due to their size. The RAVEN Toolbox support that they should be included in the model, 9 had allows for visualization of simulation results based on manually inconclusive or missing evidence, and 19 reactions should not have drawn maps. The maps are drawn in CellDesigner and each been included in the model. 5 of the 91 genes that were previously reaction is labeled with the corresponding reaction identifier in the unique to iIN800 were also added in this process. model. The map can then be imported to MATLAB and the PLOS Computational Biology | www.ploscompbiol.org 6 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox Figure 3. Overview of the genes which are unique to the automatically reconstructed model and iIN800, respectively. Saccharomyces Genome Database was used to classify the genes. Green corresponds to genes where the function is well-defined and suited for GEMs, basically enzymes involved in metabolism. Red corresponds to genes where the function is unknown, where the corresponding protein is not an enzyme or where the function is in signaling rather than metabolism. These genes should normally not be present in a GEM. Blue corresponds to genes that are putative enzymes or where the ORF is a functional enzyme in some strains but not in others. As can be seen, the automatically reconstructed model has both a larger number of unique genes and a larger proportion of enzymes compared to the published model. For iIN800 some enzymatic genes are further classified as ‘‘polymer’’, ‘‘lipid’’ or ‘‘membrane’’. These are parts of metabolism where an automatically generated model from KEGG would have particular drawbacks compared to a manually reconstructed model. ‘‘Polymer’’ corresponds mainly to genes involved in sugar polymer metabolism, which is an area that contains many unbalanced reactions in KEGG. Such reactions were excluded when the validation model was generated, so the corresponding genes could not be included. The same holds for ‘‘lipid’’, where the reactions contain many general metabolites. This also results in excluded reactions. ‘‘Membrane’’ corresponds to reactions which depend on one metabolite but in two different compartments. This compartmentalization information is absent in KEGG so the equation becomes incorrect and it is therefore excluded. doi:10.1371/journal.pcbi.1002980.g003 reactions are colored according to the change in the corresponding and A. niger which have less sequence homologues of 576 and 563, flux between simulation conditions. Gene expression data can be respectively. Upon completion of the similarity searching, the incorporated in the map to illustrate the correlation between flux results suggest that 1143 genes in P. chrysogenum could be assigned and gene expression. This will extraordinarily facilitate the as orthologous metabolic genes from the three Aspergillus species comparison and interpretation of flux distributions found for used for comparison. The large number of metabolic orthologues different environmental conditions. The resulting map is exported indicates that the existing GEMs for closely related species could as a pdf-file. Figure 4 show a close up on penicillin metabolism in be a sound foundation upon which to reconstruct the new model. the peroxisome overlaid on the full P. chrysogenum map (see following section for details). Reconstruction and comparative analysis of the P. chrysogenum metabolic network Comparative genomics of template species Using the RAVEN Toolbox the metabolic network of P. In order to assign metabolic functions to the genes present in the chrysogenum metabolic network was reconstructed. The metabolic P. chrysogenum genome, sequence alignment analysis was per- network comprises 1471 unique metabolic reactions in four sub- formed. Three fungi from the Aspergillus genus (A. oryzae, A. niger cellular compartments; extracellular, cytosolic, mitochondrial, and and A. nidulans) were selected for sequence comparison based on peroxisomal (Table 3). 1006 ORFs are associated to the reactions, 89 being closely related fungi outside of the Penicillium genus and on of which participate in one of 35 protein complexes. In parallel to the having previously reconstructed GEMs (see Figure S1). Table 2 automatic reconstruction, an extensive literature study was per- shows some genome characteristics of the Aspergilli in comparison formed. In total 440 cited articles provide experimental evidence for with P. chrysogenum. Initially pairwise comparison was done by the majority of the reactions. All model components were extensively similarity searching of the protein sequences of P. chrysogenum annotated to adhere to the MIRIAM standard for biological models against the protein sequences known to be involved in the [41]. The model was validated with respect to 76 important metabolism of the three Aspergillus species as described in the metabolic functions (the supplementary file simulations.xls is an Methods section. With a chosen threshold of the E-value, identity, input file to checkTasks, which was used to performed the validation). and alignment length, a list of inferred metabolic functions was There are 30 reactions in the model which cannot carry flux if all generated. The results are summarized in Table 2. Pairwise uptakes are allowed, i.e. dead-end reactions (see Table S7). comparison shows that A. oryzae has the highest number of sequence homologues of proteins with metabolic functions with P. According to the naming conventions for metabolic networks the chrysogenum (915 sequences). This result suggests that metabolism of presented model is denoted as iAL1006 [42]. Table 3 shows the A. oryzae is probably closer related to P. chrysogenum than A. nidulans division of model elements between the four compartments. PLOS Computational Biology | www.ploscompbiol.org 7 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox Figure 4. Example of the visualization capabilities of the RAVEN Toolbox. The figure shows a small section of the Penicillium metabolic map, depicting peroxisomal penicillin metabolism, superimposed on the full map. Rectangles correspond to reactions and ellipses correspond to metabolites. The broad yellow line represents the peroxisomal membrane. Reactions are colored based on the log-fold change in flux between a reference and a test case, where green represents a higher flux in the test case and red a lower flux. The positive direction of reversible reactions (defined as from left to right in the model equations) is indicated by a red arrow head. For reactions carrying flux in any of the simulated cases, the flux values are printed in the reaction box. The small squares to the right of some of the reactions correspond to the log-fold change of transcript levels of the genes associated to that reaction. The gene-reaction relation is retrieved from the model structure and not implicitly specified in the CellDesigner map. doi:10.1371/journal.pcbi.1002980.g004 Table 2. Comparison of genome characteristics and metabolic function assignment between P. chrysogenum and three Aspergillus species. Features ANi AO AN PC Genome size (Mb) 30.1 37.2 34.9 32.2 Number of chromosomes/supercontigs 88849 Number of total protein sequences 10 560 12 074 11 197 12 811 Functional assignments Pairwise comparison ANi and PC AO and PC AN and PC PC Number of protein sequence orthologues 5749 5614 5632 - Number of metabolic orthologues based on COG 1316 1471 1313 2330 Number of metabolic orthologues based on GEMs 576 915 563 1143 ANi: A. nidulans, AO: A. oryzae, AN: A. niger, PC: P. chrysogenum. Proteins were regarded as orthologues if E-value ,1e-30, identity .40%, sequence coverage .50%, and alignment length .200 amino acids. Described as being present in the functional category of metabolism based on the COG database [61]. Described as being present in the corresponding previously published genome-scale metabolic model; A. nidulans iHD666 [52]; A. niger iMA871 [53]; A. oryzae iWV1314 [54]. doi:10.1371/journal.pcbi.1002980.t002 PLOS Computational Biology | www.ploscompbiol.org 8 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox chrysogenum network compared to those of other fungal networks Table 3. Network characteristics of the reconstructed is available in Table S8. metabolic network of P. chrysogenum. Biomass composition and parameter fitting Growth is described as production of biomass, which in turn is ORFs 1006 regarded as drain of the macromolecules and building blocks that EC-numbers 627 constitute the cellular components. The demand of each Metabolites 1235 component is estimated based on published data on the biomass Extracellular metabolites 160 composition. The main components and their content within the Cytosolic metabolites 728 biomass are listed in Table 4 (see also Table S9 for a detailed description). The cost of biomass production does not only include Mitochondrial metabolites 242 synthesis of precursors and polymerization of macromolecules, but Peroxisomal metabolites 105 also factors such as maintaining turgor pressure, transport costs, Reactions 1471 protein turnover, and membrane leakage. These costs are Extracellular reactions 175 summarized as an ATP requirement for non-growth associated Cytosolic reactions 835 maintenance, m , and for growth associated maintenance, ATP Mitochondrial reactions 324 K , i.e. ATP costs not directly associated with biomass xATP synthesis but associated with cell growth (can be maintenance of Peroxisomal reactions 137 membrane potentials across an expanding cell). These parameters Exchange metabolites are not included. were determined by linear regression to glucose-limited chemostat Exchange reactions are not included. Transport reactions from the cytosol to experiments in the presence of phenoxyacetic acid (POA) [43]. any other compartment are included in the count for that compartment, i.e. The values were hereby estimated to be 4.14 mmol ATP/g DW/h mitochondrial transport reactions are regarded as mitochondrial reactions. for m and 104 mmol ATP/g DW for K . The growth- doi:10.1371/journal.pcbi.1002980.t003 ATP xATP associated ATP cost is significantly higher than for the template organisms (64 mmol ATP/g DW/h in A. niger iMA871). This could Figure 5 summarizes the literature support for the reactions in possibly be an effect of the presence of phenoxyacetic acid, which the model and shows a classification of the ORFs in the model is added to the fermentation medium under industrial penicillin based on the KEGG pathways. The full list of reactions, producing conditions. It is believed that phenoxyacetic acid, being metabolites, and genes are supplied in Microsoft Excel format a lipophilic weak acid, acts as a proton un-coupler which would and SBML format in Dataset S1. Both these formats are manifest itself as a high ATP maintenance cost [44]. The P/O compatible with the RAVEN Toolbox. To illustrate the metabolic ratio is fitted by assigning the number of cytosolic protons needed network, and to aid in interpretation of gene expression data and to synthesize one ATP by the F F -ATPase. This is a small 0 1 simulation results, a map of the full model was drawn in simplification since the number of protons pumped across the CellDesigner and annotated so as to be compatible with the mitochondrial membrane might also differ between organisms. visualization functions in the RAVEN Toolbox. The CellDesigner This parameter was estimated to 3.75 (3.88 in A. niger iMA871). file is available in Dataset S1. Even though the map is drawn for Figure S2 shows the agreement of model simulations with Penicillium metabolism it can be used as a template for generation experimental fermentation data after parameter fitting. of maps for other organisms as well. Lastly, the P. chrysogenum GEM has also been added to the model repository in the BioMet Simulations and integrative data analysis of penicillin Toolbox, which allows for a variety of analyses and simulations to biosynthesis be carried out. A genome-scale metabolic model is a powerful tool that can be used for exploring the metabolic capabilities of the cell, as well as Comparison of fungal metabolic networks being used as a scaffold for integrative data analysis. Here we To evaluate the similarity between the reconstructed network present two case studies to illustrate the use of the reconstructed P. and the template networks, the networks were compared with chrysogenum model. The first case is a study of penicillin yields and respect to identical reactions and involved metabolites. Only A. in particular the relative importance of ATP and NADPH oryzae iWV1314 and A. niger iMA871 were used in the comparison provision during penicillin production. In the second study we since only a small number of reaction were inferred from A. show how the model can be used to integrate fermentation data nidulans iHD666. Figure 6 illustrates the results. 534 reactions were with transcriptome data using a recently published sampling unique to iAL1006. The large discrepancy between the models is algorithm to aid in the interpretation of high-throughput data [4]. primarily because of differences in how lipid metabolism is Penicillin yields. Penicillin production is associated with an formulated and due to differences in localization. There are also increased requirement of energy in the form of ATP; in the differences in how reactions catalyzed by protein complexes are condensation of the three precursor amino acids to form the described, where one reaction for each subunit is formulated tripeptide ACV; in the reduction of sulfate; and when a side chain rather than lumping the reactions. The difference in metabolic (the precursor molecule which is supplied to the media and which capabilities between the models is therefore smaller than what is differs depending on the type of penicillin produced) is activated by indicated by the Venn diagrams (Figure 6). The unique ligation to coenzyme A. Penicillin production is also associated with capabilities of the P. chrysogenum model are mainly in penicillin a large requirement of NADPH; primarily needed for the reduction metabolism and transport (data not shown). In general, the of sulfate but also in the biosynthesis of valine and homoserine from reactions that were inferred from A. oryzae but not from A. niger are a-ketobutyrate. Elucidating the impact increased ATP require- predominantly involved in co-factor synthesis and in sugar ments have compared to the NADPH requirements is useful when polymer metabolism. The reactions inferred from A. niger choosing among possible metabolic engineering strategies. iMA871 but not from A. oryzae iWV1314 are mainly involved in Different types of penicillin can be produced by changing the lipid metabolism. The key statistics of the reconstructed P. side chain that is supplied to the medium (e.g. supplementation of PLOS Computational Biology | www.ploscompbiol.org 9 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox Figure 5. Evidence level for the P. chrysogenum metabolic network. A) Properties of the reconstructed network. The top bar shows the support for the 1471 unique reactions (not counting exchange reactions) sorted by the type of evidence. The bottom bar shows the orphan reactions; reactions inferred without supporting ORFs or literature references. B) ORF classification. The ORFs in the model are classified into broad groups based on KEGG classification. doi:10.1371/journal.pcbi.1002980.g005 phenylacetic acid result in penicillin G and supplementation of that of NADPH consumption in the sulfate reduction. The shadow phenoxyacetic acid result in penicillin V). However, this has no prices (how much the penicillin production can increase if the impact on the yield and it is therefore not necessary to specify the availability of a metabolite were to increase by a small amount) type of penicillin being produced for theoretical evaluations. The were calculated to be 0.015 mol penicillin/mol ATP, 0.040 mol maximum theoretical yield of penicillin on glucose with sulfate as penicillin/mol NADPH, and 0.037 mol penicillin/mol NADH. the sulfur source was calculated to be 0.42 mol penicillin/mol NADPH and NADH are similar when it comes to energy glucose using the reconstructed genome-scale metabolic model. content, but have different roles in the metabolism, where This is in agreement with what has previously been published [45]. NADPH serves primarily anabolic roles and NADH primarily If the sulfur source is sulfite the maximal theoretical yield is found catabolic roles. NADPH is mainly produced in the pentose to be 0.45 mol penicillin/mol glucose and if it is hydrogen sulfide phosphate pathway, which makes NADPH somewhat more it is 0.51 mol penicillin/mol glucose. The difference between using energetically expensive to regenerate compared to NADH. In sulfite and hydrogen sulfide is relatively large and can be attributed order to investigate the relative importance of NADH and to the differences in NADPH cost (3 NADPH are consumed in the NADPH an artificial reaction was included that allowed for sulfite reduction to hydrogen sulfide). This points to the production of NADPH from NADH to simulate a potential importance of NADPH availability for penicillin production. To increase of the NADPH availability. Simulations were then carried investigate the effect of ATP an artificial reaction was included out maximizing first for growth and then for penicillin production. that allowed for ATP production from ADP without any energetic The resulting flux through the artificial reaction was 8.5 times costs. This resulted in a yield of 0.52 mol penicillin/mol glucose, larger when maximizing for penicillin than when maximizing for using sulfate as the sulfur source. The conclusion is that ATP growth. This demonstrates that the cells will have a much higher availability has a relatively small effect on the yield, comparable to NADPH demand at high penicillin yields compared to normal Figure 6. Venn diagrams of model statistics for the template models A. oryzae iWV1314 and A. niger iMA871 and the P. chrysogenum iAL1006 model. A) The number of chemically distinct metabolites shared and specific for the three models, not counting presence in multiple compartments. B) The number of unique reactions shared and specific for the three models. The overlap with A. nidulans iHD666 is not shown here. doi:10.1371/journal.pcbi.1002980.g006 PLOS Computational Biology | www.ploscompbiol.org 10 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox the corresponding model IDs. Reactions that were identified as Table 4. Biomass composition of P. chrysogenum. probably being transcriptionally controlled and up-regulated are highlighted. In addition, the Reporter Metabolites algorithm was used to identify metabolites around which significant transcriptional Components Content (g/g DW) changes occurred [47]. These metabolites are highlighted in Figure 7 Protein 0.45 as well (see Table S11 for a full list of reporter metabolites). RNA 0.08 As can be seen in Figure 7, a large proportion of the reactions DNA 0.01 identified as being a transcriptionally controlled are directly involved in penicillin metabolism (15 out of 38). This indicates that Lipids 0.05 the capabilities of the industrial strain to produce penicillin to a Phospholipids 0.035 large extent depend on the reactions closely related to penicillin Sterolesters 0.010 metabolism, rather than more peripheral effects. Among these Triacylglycerides 0.005 reactions are many of the reactions responsible for the synthesis of Carbohydrates 0.25 the amino acids that are precursors for ACV as well as the two penicillin producing reactions isopenicillin N synthase and ACV Cell wall 0.22 synthase, which is consistent with a study on the gene copy- Glycogen 0.03 number effect on penicillin production [48]. The phenylacetate:- Soluble pool 0.08 CoA ligase is high ranking but the acyl-CoA:isopenicillin N Amino acids 0.04 acyltransferase is absent, which is consistent with measurements of Nucleotides 0.02 high activities of this enzyme and the low flux control estimated for Total 0.90 this enzyme [49,50]. Several of the reactions involved in sulfate reduction are present as well as the sulfate permease. It is 8% of the dry weight is constituted by ash [43]. The remaining 2% are other interesting to note that none of the reactions in the pentose soluble metabolites. phosphate pathway are identified even though there is an doi:10.1371/journal.pcbi.1002980.t004 increased demand for NADPH. We also found that the pathway from a-ketobutyrate to growth conditions. Redirecting a higher flux through the pentose succinate is identified to have both increased flux and increased phosphate pathway and/or introducing NADH-dependent versions gene expression. a-ketobutyrate is a by-product of cysteine of NADPH-consuming enzymes could therefore be potential production via the transsulfuration pathway, and it is used for metabolic engineering strategies for achieving higher penicillin yields. isoleucine biosynthesis. Under normal growth conditions the For the direct identification of possible metabolic engineering demand for cysteine is less than that for isoleucine, meaning that targets a gene deletion analysis was performed by searching for sets of all a-ketobutyrate is converted into isoleucine. However, during gene deletions that result in an increased yield of penicillin, and which high-level penicillin production the cysteine production far would stoichiometrically couple penicillin production to growth. This exceeds the need for isoleucine, requiring an alternative route was performed using FBA, and combinations of up to three gene for a-ketobutyrate consumption. This route involves the decar- deletions were evaluated (MoMA was also applied and gave similar boxylation of a-ketobutyrate to yield propionyl-CoA, which then results). The only targets which could be identified were the deletion goes into the methylcitrate pathway, eventually resulting in of any of the genes responsible for breakdown of phenylacetic acid succinate [45]. Several of the reactions in this pathway are (homogentisate 1,2-dioxygenase, maleylacetoacetate isomerase, or identified as transcriptionally controlled by the algorithm (2- fumarylacetoacetase). Deletion of any of these genes resulted in a methylcitrate synthase, 2-methylcitrate dehydratase, 2-methyliso- 21% increase in penicillin production when maximizing for growth. citrate dehydratase, and methylisocitrate lyase). This finding Identification of transcriptionally regulated metabolic strongly supports that the transsulfuration pathway is the bottlenecks. The metabolism of cells is redundant in the sense dominating pathway for cysteine biosynthesis, even though the that different sets of metabolic reactions can be used to generate enzymes for the energetically more efficient direct sulfhydrylation the same net phenotype. A recently developed method aims at pathway have been identified in P. chrysogenum [51]. finding potential metabolic engineering targets by identifying genes that are differentially expressed between different cultivation Discussion conditions and where the corresponding reactions exhibit signif- icantly changed fluxes for the same conditions [4]. Changes in The RAVEN Toolbox, a software suite for semi-automated expression level of such genes are then assumed to be likely to reconstruction and simulation of genome-scale metabolic models result in altered fluxes. The algorithm finds these transcriptionally was developed. The RAVEN Toolbox is the first software that regulated reactions by random sampling of the solution space, contains methods both for model reconstruction and for a wide after which it compares the statistics of the sampling with the variety of simulation approaches. A visualization feature for statistic of the mRNA expression. Here we applied this method to simulation results and a feature that allows the user to manipulate compare the high producing industrial strain DS17690, which has metabolic models and set simulation parameters via Microsoft Excel been developed by DSM, and the low producing reference strain are provided in order to make the software easy to use. The RAVEN Wis 54-1255 (see Methods for details) [46]. Toolbox was evaluated for its ability to reconstruct GEMs by A total of 58 fluxes were found to be significantly changed between generating a model for S. cerevisiae. The reconstructed model the high and low production strains (p,0.05) and 612 genes were compares well with a manually reconstructed model. This demon- differentially expressed (p,0.005). Out of those, 36 reactions were strates that the RAVEN Toolbox is useful for reconstruction of novel identified as having significantly higher flux and up-regulated genes models, in particular eukaryotic models, due to its feature for (see Table S10), i.e. they are likely to have transcriptional regulation automatic assignment of sub-cellular localization. We used the of their fluxes. Figure 7 shows some of the most important reactions RAVEN Toolbox to reconstruct a GEM for P. chrysogenum by using in penicillin biosynthesis together with the responsible enzymes and three models for closely related fungal species. Extensive manual PLOS Computational Biology | www.ploscompbiol.org 11 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox Figure 7. Integrative analysis of a high and a low producing strain. Depicts synthesis pathways of penicillin and important precursors. Green boxes correspond to reactions identified as being transcriptionally controlled and up-regulated by the algorithm (see text). Metabolites around which significant transcriptional changes occur compared to a low producing strain are colored red. SC: side chain (e. g. the precursor molecule phenylacetic acid). The biosynthesis of penicillin starts with the condensation of the three amino acids a-aminoadipate (an intermediate in the L- lysine biosynthesis pathway), L-cystein, and L-valine to form the tripeptide ACV. ACV is further converted to isopenicillin N. For the industrially relevant types of penicillin a side-chain is supplied to the media. This side-chain is activated by ligation to coenzyme A. In the last step of penicillin biosynthesis an acyl transferase exchanges the a-aminoadipate moiety of isopenicillin N with the side-chain, thereby generating penicillin and regenerating a-aminoadipate. Since L-cystein is a sulfur-containing amino acid penicillin production is also tightly associated with sulfur metabolism. The corresponding model IDs for the enzymes are indicated within parentheses. [1] homocitrate synthase (r0683); [2] homocitrate dehydrase (r0684); [3] homoaconitate hydrase (r0685); [4] homoisocitrate dehydrogenase (r0688); [5] a-aminoadipate aminotransferase (r0689); [6] homoserine transacetylase (r0600); [7] O-acetylhomoserine sulfhydrylase (r0601); [8] cystathione-b-synthase (r0632); [9] cystathione-c-lyase (r0606); [10] acetate CoA ligase (r0025); [11] acetolactate synthase (r0465); [12] ketol-acid reductoisomerase (r0653); [13] dihydroxy acid dehydrase (r0656); [14] branched chain amino acid transferase (r0648); [15] ACV synthase (r0814); [16] isopenicillin N synthase (r0812); [17] acyl CoA ligase (side chain dependent, reaction is for phenylacetate CoA ligase) (r0747); [18] isopenicillin N N-acyltransferase (r0813); [19] sulfate permease (r1408); [20] sulfate adenyl transferase (r1151); [21] adenyl sulfate kinase (r1147); [22] phosphoadenyl sulfate reductase (r1148); [23] sulfite reductase (r1149); [24] thioredoxin reductase (r0419); [25] 39(29),59-bisphosphate nucleotidase (r1150). doi:10.1371/journal.pcbi.1002980.g007 validation of the model was performed; both to validate the manual curation, and an extensive bibliomic survey. Figure 8 reconstruction method and to ensure a high-quality model. The gives an overview of the whole reconstruction process. resulting P. chrysogenum model consists of 1471 reactions, 1235 metabolites and 1006 genes. 440 cited articles provide experimental Inferring reactions based on protein homology evidence for the majority of the reactions. Considerable efforts were Three GEMs for other filamentous fungi, A. nidulans iHD666 spent on standardizing and annotating the template models in order [52], A. niger iMA871 [53], and A. oryzae iWV1314 [54], were used to adhere to MIRIAM standards. The standardized template models as template models for the reconstruction of a P. chrysogenum model. and the reconstructed P. chrysogenum model are available through the Efforts were taken in order to standardize the template models to BioMet Toolbox. This collection of fungal models, together with the facilitate the automatic reconstruction. This standardization complementary method of generating metabolic networks based on primarily involved metabolite naming, but also how to represent the KEGG database, constitutes an excellent platform for the more complex aspects of metabolism such as polymers and lipids. As reconstruction of metabolic networks for other eukaryotic organisms. part of the standardization effort a large majority of the metabolites were assigned database identifiers and chemical structure informa- Methods tion. This annotation step allowed for verification that all reactions The P. chrysogenum metabolic network was reconstructed based were elementally balanced, which in turn led to a number of on a combination of automated reconstruction approaches, inconsistencies in the template models being corrected. The revised PLOS Computational Biology | www.ploscompbiol.org 12 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox Figure 8. Overview of the iAL1006 reconstruction process. doi:10.1371/journal.pcbi.1002980.g008 models for the three Aspergillus species are available as up-dates in metabolic pathways that were not included based on the template the BioMet Toolbox (http://www.sysbio.se/BioMet) [28]. models. No reactions were included based solely on presence in A draft GEM for P. chrysogenum was then constructed based on the KEGG model, and gene assignments were only included after bidirectional best hits of BLASTp between the template model careful manual validation against the NCBI RefSeq database. proteins and their orthologues in P. chrysogenum using the RAVEN Gaps in the draft metabolic network were identified using the Toolbox. Proteins were regarded as orthologues if E-value ,1e- gap finding capabilities of the RAVEN Toolbox (Figure 8). The 30, identity .40%, sequence coverage (.50%) and alignment initial network was rather disconnected and biomass production length (.200 amino acids). from glucose was not possible. Firstly, the software was used to The protein sequences of P. chrysogenum Wisconsin 54-1255 identify which metabolites had to be connected in order to (annotation, version 1) were obtained from the EMBL database produce biomass. This resulted in the addition of some sponta- (http://www.ebi.ac.uk/embl/). The protein sequences of A. nidulans neous reactions from the template models. The second step was to FGSC A4 (annotation, version 4) were taken from the Broad ensure that as many metabolites as possible could be produced. Institute database (http://www.broadinstitute.org/annotation/ When non-connected metabolites were identified, the KEGG genome/aspergillus_group). The protein sequences of A. oryzae model was queried for candidate reactions which could connect RIB40 (annotation, version 1) were taken from the DOGAN that metabolite. A targeted literature search was then conducted to database (http://www.bio.nite.go.jp/dogan/project/view/AO). find evidence for the presence of such a reaction. In only few cases this resulted in the addition of transport reactions based solely on The protein sequences of A. niger ATCC1015 (annotation, version 3) were taken from the JGI database (http://genome.jgi-psf.org/ connectivity issues (,2%, see Figure 5). The final step was to use the RAVEN Toolbox to identify reactions that could not carry a Aspni5/Aspni5.home.html). flux during growth on any of the available carbon sources. There are, however, sets of reactions that are included in the model even Gap filling though they cannot currently carry a flux. One such example is the The first draft model based on homology to template models synthesis and loading of tRNA, which is included to allow for contained gaps due to incorrect annotation in the template models possible future extension of the model to cover protein synthesis. and lacked reactions in parts of metabolism that were unique to P. chrysogenum (Figure 8). Therefore, another draft model was generated from KEGG using the RAVEN Toolbox. An E-value Compartmentalization and transport ,1e-50 was used as cut off in the gene assignment. This model was The model has four compartments: extracellular space, cyto- used for filling gaps in the draft network and for suggesting sol, mitochondrion, and peroxisome. Reactions with unknown PLOS Computational Biology | www.ploscompbiol.org 13 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox localization, or where the real localization is not represented by Supporting Information one of the compartments in the model, were assigned to the Dataset S1 The iAL1006 genome-scale model of P. chrysogenum cytosol. Enzymes reported to be present in the cell wall were in SBML and Excel formats, together with a metabolic map for assigned to the extracellular space, and those present in the visualization and a task list for model validation. mitochondrial membrane were mainly assigned to the mitochon- (ZIP) dria. The peroxisome was included primarily because of its role in penicillin metabolism and b-oxidation of fatty acids. Transport Figure S1 Proteome comparison of genomes in Fungi. ALR (the reactions between compartments were inferred mainly from the ratio of alignment length to query sequence length): .0.50, template fungal models and backed up with literature evidence. identity: .0.40. The red shades refer to protein homology that can However, there were situations where a transport reaction had to found within a genome (paralog). The green shades refer to protein homology that can found between two genomes (ortholog). be included in order to have a functional network, even when no literature evidence could be found. For enzymes reported to have (PDF) isoenzymes in several compartments, the ORF assignments to Figure S2 Agreement of model simulations with experimental each compartment were based on localization predictions from fermentation data. Data from glucose-limited chemostat with defined CELLO [36] and pTarget [55]. medium containing glucose, inorganic salts and phenoxyacetate. (PDF) Simulations Table S1 Reactions which were excluded from the general Unless otherwise specified, simulations were carried out using KEGG model after running removeBadRxns. 72 reactions were unlimited uptake of oxygen, phosphate, sulfate, NH3, thiamin and unbalanced, general or polymer reactions and were therefore pimelate. The carbon source was glucose. Excretion of all correctly removed. 7 reactions were correct in KEGG, but were exchange metabolites was allowed. Biosynthesis of L-cysteine removed because they lacked metabolite composition (it is a setting was only allowed through the transsulfuration pathway. Since the in removeBadRxns whether it is allowed to remove such reactions). energy content of NADH and NADPH is similar it is possible that (PDF) cycles convert one into the other. These cycles normally take the form of reversible reactions that can utilize either NADH or Table S2 Comparison of an automatically reconstructed model for S. cerevisiae to a published model of the same organism (iIN800) in NADPH. Since such futile cycles make it difficult to study terms of included genes. The table shows the genes that are unique NADPH/NADH metabolism using GEMs, and since they are to either the automatically reconstructed or the manually probably not active in the cell, they were identified and blocked in reconstructed model, and a classification of the genes into groups analogy to what has been done in previous models [10]. The way that reflect how well suited they are for being included in a GEM. by which they were identified was minimizing/maximizing for the Genes labeled as ‘‘enzymatic’’ should be included, while all other flux through an artificial reaction NADH+NADP(+), = . groups should probably be excluded. For iIN800 some enzymatic NAD(+)+NADPH while not allowing for uptake of any carbon genes are further classified as ‘‘polymer’’, ‘‘lipid’’ or ‘‘membrane’’. source. Reactions were then identified and deleted until this These are parts of metabolism where an automatically generated reaction could no longer carry a flux. The following reactions were model from KEGG would have particular drawbacks compared to deleted: D-mannitol:NAD+ 2-oxidoreductase (r0181); ethanol:- a manually reconstructed model. ‘‘Polymer’’ corresponds mainly to NADP+ oxidoreductase (r0019); ethanol:NAD+ oxidoreductase genes involved in sugar polymer metabolism, which is an area that (r0020); (S)-3-hydroxybutanoyl-CoA:NADP+ oxidoreductase contains many unbalanced reactions in KEGG. Such reactions (r0081). None of these reactions can be expected to be active were excluded in the validation, so the corresponding genes could during the studied conditions. Deletion of D-mannitol:NAD+ 2- not be included. The same is true for ‘‘lipid’’, where the reactions oxidoreductase does not inactivate the NADPH regenerating contain many general metabolites, which also results in excluded mannitol cycle, which has a role in NADPH regeneration in many reactions. ‘‘Membrane’’ corresponds to reactions which depend on fungal species [56]. any one metabolite in different compartments. This compartmen- The penicillin yields were calculated by setting the glucose talization information is absent in KEGG so such a reaction would uptake rate to 1.0 mmol/gDW/h and maximizing for penicillin read, for example, A+B=.A+C. ‘‘A’’ here might mean ‘‘A(cyto- production. Free ATP was simulated by including an artificial solic)’’ and ‘‘A(mitochondrial)’’, but since that information is reaction in the form ADP+Pi = .ATP+H2O. missing, the equation becomes incorrect and it is therefore excluded. ‘‘Signaling’’ corresponds to proteins which are primarily involved in Integrative analysis signaling, even though they might have an enzymatic capability. A random sampling algorithm was applied in order to identify (PDF) transcriptionally regulated metabolic bottlenecks [4]. Flux data and Table S3 Metabolites which could be synthesized in the gene expression levels for aerobic, glucose-limited chemostat fermen- automatically reconstructed S. cerevisiae model from minimal media tation of DS17690 and Wis 54-1255 were used as input to the (glucose, phosphate, sulfate, NH3, oxygen, 4-aminobenzoate, algorithm [57]. The exchange fluxes were fitted to the reported values riboflavin, thiamine, biotin, folate, and nicotinate). Uptake of the using a quadratic fitting. 5000 sampling iterations were performed for carriers carnitine and acyl-carrier protein was allowed for modeling each of the two strains. The expression data set of the study were purposes (many compounds are bound to them and therefore net retrieved from GEO database (GSE9825) as CEL format then synthesis of these compounds is not possible without them). normalized together with PLIER workflow (http://media.affymetrix. (PDF) com/support/technical/technotes/plier_technote.pdf). Two way AN- OVA were employed to evaluate the differentially expressed genes Table S4 New metabolites which could be synthesized in the with respect to the strain (DS17690 and Wis 54-1255) with multiple automatically reconstructed S. cerevisiae model from minimal media correction following [58]. The Reporter algorithm [47] was employed after gap-filling. These metabolites were all present in the model to integrate the transcriptome data with the reconstructed GEM to before the addition of new reactions. identify key metabolites in the network. (PDF) PLOS Computational Biology | www.ploscompbiol.org 14 March 2013 | Volume 9 | Issue 3 | e1002980 The RAVEN Toolbox Table S5 Reactions which were added to the automatically Table S10 Reactions with significantly higher flux in DS17690 reconstructed S. cerevisiae model by fillGaps. Out of the 45 added compared to Wis 54-1255 where the corresponding genes are also reactions 17 has evidence to support that they should be included up-regulated. Ranked by significance (p,0.05). (PDF) in the model, 9 has inconclusive of missing evidence, and 19 should not have been included in the model. Table S11 Reporter metabolites when comparing the DS17690 (PDF) and Wis 54-1255 strains. Ranked by significance. Top 40 best scoring metabolites are shown. Table S6 Genes where their corresponding reactions were (PDF) localized to the mitochondria after running predictLocalization (transport cost = 0.1). The color indicates whether the gene Acknowledgments product is mitochondrial in SGD, where green means that it does, yellow that it is unclear, and red that it does not. We thank Timo Hardiman and Rudolf Mitterbauer at Sandoz for valuable (PDF) feedback throughout the project, and Sinisa Bratulic for helping with the generation of the CellDesigner map. Table S7 Reactions which cannot carry flux even when all uptake reactions are unconstrained. Author Contributions (PDF) Performed the reconstruction: RA LL. Bioinformatics analyses: WV IN. Table S8 Comparison of metabolic models. Developed the sub-cellular localization algorithm: RA SS. Wrote the (PDF) software and performed the simulations: RA. Supervised the work: IN JN. Conceived and designed the experiments: RA JN. 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Ostergaard S, Theilgaard HBA, Nielsen J (1998) Identification and purification of O-acetyl-L-serine sulphhydrylase in Penicillium chrysogenum. Applied Microbiology and Biotechnology 50: 663–668. PLOS Computational Biology | www.ploscompbiol.org 16 March 2013 | Volume 9 | Issue 3 | e1002980 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS Computational Biology Public Library of Science (PLoS) Journal

The RAVEN Toolbox and Its Use for Generating a Genome-scale Metabolic Model for Penicillium chrysogenum

PLoS Computational Biology , Volume 9 (3): e1002980 – Mar 21, 2013

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