A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory

  • Juan Nogales1,

    Affiliated with

    • Bernhard Ø Palsson2Email author and

      Affiliated with

      • Ines Thiele2, 3

        Affiliated with

        BMC Systems Biology20082:79

        DOI: 10.1186/1752-0509-2-79

        Received: 05 March 2008

        Accepted: 16 September 2008

        Published: 16 September 2008

        Abstract

        Background

        Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions.

        Results

        We present a genome-scale reconstruction of P. putida KT2440's metabolism, iJN746, which was constructed based on genomic, biochemical, and physiological information. This manually-curated reconstruction accounts for 746 genes, 950 reactions, and 911 metabolites. iJN746 captures biotechnologically relevant pathways, including polyhydroxyalkanoate synthesis and catabolic pathways of aromatic compounds (e.g., toluene, benzoate, phenylacetate, nicotinate), not described in other metabolic reconstructions or biochemical databases. The predictive potential of iJN746 was validated using experimental data including growth performance and gene deletion studies. Furthermore, in silico growth on toluene was found to be oxygen-limited, suggesting the existence of oxygen-efficient pathways not yet annotated in P. putida's genome. Moreover, we evaluated the production efficiency of polyhydroxyalkanoates from various carbon sources and found fatty acids as the most prominent candidates, as expected.

        Conclusion

        Here we presented the first genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. Taken together, this work illustrates the utility of iJN746 as i) a knowledge-base, ii) a discovery tool, and iii) an engineering platform to explore P. putida's potential in bioremediation and bioplastic production.

        Background

        Pseudomonas putida is a non-pathogenic member of rRNA group I of the genus Pseudomonas that colonizes many different environments and is well known for its broad metabolic versatility and genetic plasticity [1, 2]. P. putida KT2440 is a TOL plasmid cured, spontaneous restriction deficient derivative of P. putida mt-2 [3, 4]. This strain represents the first host-vector biosafety system for cloning in gram-negative soil bacteria and hence, has been extensively used as a host for gene cloning and expression of heterologous genes [58]. Consequently, large efforts have been made in exploiting these capacities in a diverse range of biotechnological applications including i) bioremediation of contaminated areas [9, 10]; ii) quality improvement of fossil fuels, e.g., by desulphurization [11]; iii) biocatalytic production of fine chemicals [9, 1214]; iv) production of bioplastic [1517]; and v) as agents of plant growth promotion and plant pest control [18, 19].

        Since the publication of P. putida KT2440's genome [20], our knowledge about this strain has significantly increased [21] and various "-omics" data sets have become available, such as transcriptomic [22, 23], proteomic [24], and fluxomic data [25, 26]. Subsequently, some in silico analyses of its metabolic and biotechnological capacities have been published [27, 28]. However, systemic understanding of metabolic and biotechnology capabilities of P. putida KT2440 requires the construction of a more comprehensive model enabling the integration of the canonical experimental data along with genomic and high-throughput data in a hierarchical and coherent fashion [29].

        The constraint-based reconstruction and analysis (COBRA) approach is one possible modeling approach that uses stoichiometric information about biochemical transformation taking place in a target organism to construct the model. While a metabolic reconstruction is unique to the target organism one can derive many different condition-specific models from a single reconstruction. This conversion of a metabolic reconstruction of an organism into models requires the imposition of physicochemical and environmental constraints to define systems boundaries [3032]. The conversion also includes the transformation of the reaction list into a computable, mathematical matrix format. In this so-called S matrix, where S stands for stoichiometric, the rows correspond to the network metabolites and the columns to the network reactions. The coefficients of the substrates and products of each reaction are entered in the corresponding cell of the matrix. This conversion can be done automatically (e.g., using the Matlab-based COBRA toolbox [33]). Once in this format, numerous mathematical tools can be used to interrogate the metabolic network properties in silico. Many of the published mathematical tools have been reviewed [34] and encoded in Matlab format [33]. A large subset of these tools relies on linear programming (LP), a mathematical tool used to find a solution to an optimization problem (e.g., maximal possible growth rate of my metabolic network under a given set of environmental constraints). While LP-based tools are very helpful in studying reconstructed metabolic networks, some questions may better be addressed without having to choose an objective function. Those methods are called unbiased methods, in contrast to biased LP-based methods, because they identify all feasible flux distributions under the given set of environmental constraints rather than only the optimal distributions. The COBRA approach [30, 32] has been successfully used to build and analyze genome-scale in silico reconstructions for representatives of archaea (e.g.,Methanosarcina barkeri [35]), of bacteria (e.g., E. coli [36]; B. subtilis [37]; H. pylori [38]; M. tuberculosis [39, 40]; S. aureus [41, 42]; L. lactis [43]), and of eukarya (e.g., Human [44]). The numerous mathematical tools have been used for i) identification and filling of knowledge gaps (e.g. missing gene annotations [45]); ii) prediction of the outcome of adaptive evolution [4648]; iii) design of engineered production strains [49]; and iv) the understanding of topological features of metabolic networks [5053]. A recent review illustrates the variety of questions that have been addressed to E. coli's metabolic network using different biased and unbiased COBRA methods [54].

        Here, we describe a highly detailed, genome-scale, metabolic reconstruction of Pseudomonas putida KT2440. Based on the naming convention for metabolic networks [55], this genome scale reconstruction was deemed iJN746, where i stands for in silico, JN are the initials of the constructor, and 746 corresponds to the number of included metabolic genes. The reconstruction was built using the COBRA approach [30, 32] and validated using flux balance analysis (FBA, [56]). The in silico metabolic network was further evaluated by comparing i) predicted growth rate capacities in different carbon sources and ii) predicted essential genes with experimental data from P. putida KT2440 and P. aeruginosa. Finally, we show the utility of the P. putida reconstruction to analyze its biodegradative (i.e. toluene degradation) and biotechnological (i.e. bioplastic production) capacities.

        Results and discussion

        Characteristics of the metabolic reconstruction of Pseudomonas putida KT2440

        The metabolic reconstruction of P. putida KT2440, iJN746, was constructed based on its annotated genome sequence [20], primary and review publications, various genetic and biochemical databases (i.e., KEGG Database [57], PSEUDOCYC [58], and SYSTOMONAS [59]), and biochemical information found in Pseudomonas-specific [21] and biochemical textbooks.

        iJN746 accounts for 746 open reading frames (ORF), whose corresponding gene products are involved in 810 metabolic and transport reactions (Table 1). A total of 140 non-gene associated reactions were included in iJN746 based on physiological evidence in literature supporting their presence in P. putida's metabolism. Hence, the reconstruction captures a total of 950 metabolic reactions and 911 metabolites distributed over three different cellular compartments: cytoplasm, periplasm, and extracellular space. Each metabolite was placed in one or more of these compartments depending on the cellular localization of the catalyzing enzyme, and the flux across outer and inner membranes was enabled by transport reactions.
        Table 1

        Properties of metabolic reconstruction of P. putida KT2440

        Reconstruction & Organism

         

        iJN746

        P. putida

        iAF1260

        E. coli

        iYO844

        B. subtilis

        iNJ661

        M. tuberculosis

        iMO1056

        P. aeruginosa

        Protein coding genes per genome

        5,350a

        4,464b

        4,106a

        3,989a

        5669

        SKI valuec

        0.74

        55.87

        4.97

        7.84

        5.12

        Genes (% of genome)

        746 (14%)

        1260 (28%)

        844 (21%)

        661 (17%)

        1056 (18,6%)

        Reactions

        950

        2077

        1020

        939

        883

        Gene-reaction associated

        810

        1919

        904

        723

        839

        Non-gene- associated network reaction (% of network reactions)

        140 (17%)

        158 (8%)

        116 (13%)

        116 (16%)

        44 (5%)

        Exchange reactions

        90

        304

        225

        88

        -

        Metabolites

        911

        1039

        988

        828

        -

        Properties of metabolic reconstruction of P. putida KT2440 were compared with recently published metabolic reconstructions of E. coli MG1655 (iAF1260 [36]), B. subtilis (iYO844 [37]), and M. tuberculosis H37Rv (iNJ661 [39]) and P. aeruginosa (iMO1056 [64]). a taken from KEGG [57]; b based on Riley et al. [98]; c Species Knowledge Index (SKI) was calculated as described in [65].

        The reactions included in iJN746 were divided into 55 specific pathways, or subsystems, based on their functional role (Figure 1A). In general, the transport subsystem was found to be the subsystem with the highest number of gene-associated reactions, highlighting the importance of cellular transport for P. putida. This observation agrees well with the known lifestyle of P. putida [28] and successfully reflects the fact that approx. 12% of P. putida genome encodes for transport-associated gene products [20]. Reactions related to amino acid metabolism were also found to be very important since the de novo synthesis pathways for all 20 amino acids are present in P. putida's genome [20]. Moreover, P. putida is known for its capability to utilize many amino acids as a carbon and nitrogen source [21, 60]. A third group of great importance contained reactions involved in aromatic acid degradation pathways, which reflects the physiological ability of P. putida to use many of these compounds as a carbon and energy source (see Figure 2) [27]. Furthermore, despite the absence of the TOL pathway in KT2440's genome, the plasmid genes and the corresponding reactions were included into the P. putida metabolic reconstruction since the TOL plasmid is present in the parental strain P. putida mt-2 and this paradigmatic plasmid is often used to expand P. putida KT2440's metabolic capacities [6, 12]. Finally, reactions associated with lipid metabolism constituted another important subsystem group. In fact, P. putida KT2440 can synthesize and accumulate medium-side-chain polyhydroxyalkanoates (msc-PHAs), which are lipid related polymers, from a wide range of carbon sources [17, 61]. This ability is of special interest for biotechnological purposes (reviewed in [62, 63]) and therefore, we incorporated both the msc-PHAs biosynthetic and TOL biodegradative pathways into the metabolic reconstruction (see below).
        http://static-content.springer.com/image/art%3A10.1186%2F1752-0509-2-79/MediaObjects/12918_2008_Article_236_Fig1_HTML.jpg
        Figure 1

        A. Pie chart showing the distribution ofiJN746's intracellular reactions over the different subsystems. The number of reactions per subsystem is shown and subsystems of high importance were highlighted in bold. B. Heat map of the confidence score of the different subsystems in iJN746. The 4 rows in the map represent the different confidence score (from left to right: 4, 3, 2, 1). The various colors correspond to the percentage of subsystems reactions that have the corresponding confidence score (red = 100%, blue = 0%). The confidence level was based on a scale from 1 to 4. A level, or score, of 4 corresponds to biochemical evidence for a gene product and its reaction(s); 3 represents physiological, genetic, or proteomic evidence; 2 corresponds to only sequence-based evidence for a gene product and its reaction(s); and finally a score of 1 reflects that the reaction had to be included for model functionality (e. g., production of biomass precursor).

        http://static-content.springer.com/image/art%3A10.1186%2F1752-0509-2-79/MediaObjects/12918_2008_Article_236_Fig2_HTML.jpg
        Figure 2

        General depiction of the aromatic compound degradation routes present iniJN746. The protocatechuate (pca genes) and catechol (cat genes) branches of the beta-ketoadipate pathway are shown as well as peripheral pathways by orange arrows. The homogentisate pathway (hmg genes) is represented by green arrows and the phenylacetate pathway (paa genes) is represented by purple arrows. The nicotinate and gallate pathways (unknown genes) are shown by green and red arrows, respectively. Finally, the Tol pathway (xyl genes from pWW0 plasmid) for toluene and xylene degradation is represented by blue arrows. The initial aromatic compounds are indicated by green circles and the central metabolic compounds for each pathway are also highlighted. A detailed list of reactions involved in aromatic acid degradation can be found in the Additional file 9.

        Every network reaction was associated with confidence scores based on the available evidence for its presence in the P. putida metabolic network (Figure 1B). For instance, reactions whose enzymes have been biochemically studied in P. putida received a confidence score of 4. If physiological or genetic knockout information was available, a score of 3 was associated with the network reaction. Reactions associated with enzymes that were only annotated in P. putida's genome but had no further experimental evidence were given a confidence score of 2. Finally, during the evaluation of the network functionality (i.e. biomass precursor production) some reactions had to be added to the network for which no genetic or experimental evidence could be found. Those reactions represent modeling hypotheses, which need further experimental validation and thus received a confidence score of 1. Upon completion, the reconstruction had an overall average confidence score of 2.83. In fact, two thirds of P. putida's metabolic pathways have been very well or well studied, while only a third of the subsystems were primarily based on the genome annotation (Figure 1B). This high level of confidence is also reflected by the number of references that lead to this metabolic reconstruction. Almost 90% of the internal reactions (844) have at least one associated citation, while a total of 176 unique primary and review publications were reviewed and incorporated into this reconstruction. Subsequently, this first genome-scale reconstruction of P. putida's metabolism represents a comprehensive knowledge base summarizing and categorizing the information currently available. The content of this knowledge base will be easily accessible through the BiGG database http://​BiGG.​ucsd.​edu.

        Comparison of scope and content of iJN746 with published metabolic networks

        The properties of iJN746 were compared with the properties of recently published reconstructions of E. coli MG1655 (iAF1260, [36]), B. subtilis (iYO844 [37]), M. tuberculosis H37Rv (iNJ661 [39]), and P. aeruginosa PAO1 (iMO1056 [64] (Table 1). We found that the percentage of included ORFs was smaller in iJN746 than in the other reconstructions. Subsequently, it can be expected that the number of metabolic functions present in P. putida is larger than currently identified in the genome annotation and literature. In fact, the number of included non-gene associated reactions was twice that of the E. coli metabolic reconstruction. Furthermore, the species knowledge index (SKI) [65], which relates the number of PubMed abstracts of an organism to its number of ORFs, was much lower for P. putida compared to the other reconstructions. In summary, this comparison indicates that the overall context coverage in iJN746 is comparable with other high-quality network reconstructions when the amount of available literature is considered.

        A metabolic reconstruction for another representative of the Pseudomonas genera was published recently [64]. A comparison of P. putida and P. aeruginosa metabolic reconstructions was performed (Table 1). In contrast to P. putida, P. aeruginosa is an opportunistic human pathogen and as such more information about its metabolism and physiology is available, which is directly reflected by a SKI value 7 times higher than that of P. putida (Table 1). As a consequence, a larger number of metabolic genes were included in the metabolic reconstruction (14% of P. putida's genome vs. 18% of P. aeruginosa's genome). Despite being close relatives, these two representatives have significant differences in lifestyle and metabolic capabilities. Subsequently, the two metabolic reconstructions have significant differences, emphasizing the importance of organism-specific reconstructions. For instance, the P. aeruginosa reconstruction contains pathways necessary for growth and production of common virulence factors, including alginate, rhamnolipids, phenezines, and quorum-sensing molecules [64], which are not present in P. putida's metabolic network. In contrast, P. aeruginosa's metabolic network does not account for pathways necessary to degrade aromatic compounds.

        iJN746's metabolic versatility

        Flux balance analysis (FBA [56]) can provide insight into the growth capabilities of the reconstructed network. Comparison of in silico growth performance with experimental data allows for the assessment of the predictive potential of the metabolic reconstruction and thus represents a valuable tool for network evaluation. Furthermore, in silico growth analysis may expand the known array of carbon-, nitrogen-, and energy sources of the reconstructed organism. In this study, the aerobic growth capabilities of iJN746 in iM9 medium substituted with different carbon sources were determined qualitatively (Table 2) and quantitatively (Table 3). The growth simulation results reflected the metabolic versatility for which P. putida is well known, with a total of 59 carbon sources enabling in silico growth when added to the iM9 minimal medium (Table 2). Furthermore, we compared the in silico growth performance on different carbon, sulfur, and nitrogen sources with phenotyping data derived from literature [see Additional file 1]. For instance, P. putida is found in terrestrial and aquatic environments around the world, with preference for the rhizosphere [21], which is especially rich in carbon sources, amino acids, organic acids, and aromatic acids derived from seeds, roots, and other plant parts [66, 67]. This niche specificity accounts for the broad carbon source usage of KT2440 and therefore, most of the known soil carbon sources were captured in iJN746 (Table 2). Of particular biotechnological importance is the ability of iJN746 to metabolize aromatic compounds, thus, representing the first metabolic reconstruction accounting for growth on these carbon sources. For example, aromatic compounds such as toluene or xylene are of special interest as they are archetypical pollutants. Subsequently, we studied the toluene degradation process using iJN746 (see below).
        Table 2

        Carbon sources enabling growth of iJN746 in iM9 mineral medium.

        Class

        Compound

        Class

        Compound

        Aromatic and related compounds

         

        Amino acids

         
         

        Protocatechuate

         

        L-Arginine

         

        Caffeate

         

        L-Aspartate

         

        Oxoadipate

         

        L-Glutamate

         

        4-Hydroxybenzoate

         

        L-Glycine

         

        Benzoate

         

        L-Histidine

         

        Catechol

         

        L-Isoleucine

         

        Coniferyl alcohol

         

        L-Leucine

         

        Ferulate

         

        L-Lysine

         

        Gallate

         

        L-Proline

         

        m-Xylene

         

        L-Serine

         

        Nicotinate

         

        L-Threonine

         

        p-Xylene

         

        L-Valine

         

        Phenylacetate

        Organics acids

         
         

        L-Phenylalanine

         

        α-Ketoglutarate

         

        Quinate

         

        Citrate

         

        p-Coumarate

         

        Fumarate

         

        Toluene

         

        Isocitrate

         

        L-Tyrosine

         

        D-Lactate

         

        Vanillin

         

        L-Lactate

         

        Vanillate

         

        Malate

        Fatty acids

          

        Succinate

         

        Acetate

        Carbohydrates

         
         

        Decanoate

         

        2-ketogluconate

         

        Dodecanoate

         

        D-Fructose

         

        Hexadecanoate

         

        D-Glucose

         

        Hexanoate

         

        D-Gluconate

         

        Octanoate

         

        D-Ribose

         

        Propionate

        Miscellaneous compounds

         
         

        Tetradecanoate

         

        4-Aminobuturate

        Polyalcohols and glycols

          

        Glycine betaine

         

        Glyceraldehyde

         

        Ornithine

         

        Glycerol

         

        Choline

         

        Glycolate

         

        Choline sulfate

        Carbon sources enabling growth of iJN746 in iM9 mineral medium. The compounds were grouped based their structural characteristics. A complete list of carbon sources tested, along with possible nitrogen and sulfur sources, as well as bibliographic support can be found in the Additional file 2.

        Table 3

        Comparison of growth performance of the in silico strain iJN746 and KT2442.

        Strain

        #x00A0;

        Carbon

        source

        μmax(h-1)

        iJN746

        μmax(h-1)

        KT2442

        Carbon Uptake rate

        (mmol gDW/h)

        O2 Uptake rate

        (mmol gDW/h)

        iJN746/KT2442

        Glucose

        0.751

        0.56a

        6.3a

        15.34d

        iJN746/KT2442

        Toluene

        0.421

        0.72b

        11.9b

        18.5c

        iJN746/KT2442

        Toluene

        0.476

        0.72

        11.9

        20.93d

        iJN746/KT2442

        Toluene

        0.7255

        0.72

        11.9

        33

        iJN746/KT2442

        Toluene

        1.262

        0.72

        11.9

        Comparison of growth performance of the in silico strain iJN746 and KT2442. The in silico growth rate was calculated in iM9 minimal medium plus glucose or toluene. Due to candidate oxygen limited growth in toluene, the in silico growth rate was calculated under different oxygen uptake rates In addition iJN746 growth in toluene as only carbon source was simulated at different oxygen uptake rates. a from [25]; b from[26]; c from [78], and d experimentally determined in this study.

        No false positive carbon, nitrogen, or sulfur sources were found in iJN746, as expected, as only exchange reactions were included in the reconstruction for metabolites, which have been reported to be taken up or secreted by P. putida KT2440. In contrast, some disagreements, such as false negatives, were observed despite a good overall agreement with the in vivo data [68] [Additional file 1]. For example, it was reported that P. putida can use L-alanine as a carbon- and nitrogen-source [68] but iJN746 cannot use this compound as a carbon or nitrogen source. This disagreement could not be resolved. In contrast, iJN746 was initially unable to use choline-O-sulphate, choline, or glycine betaine as carbon- and nitrogen-sources despite experimental evidence [69]. However, the addition of two non-gene-associated reactions, betaine-homocysteine S-methyltransferase (EC- 2.1.1.5) and dimethylglycine dehydrogenase (EC- 1.5.99.2), enabled iJN746 to use these metabolites as carbon- and nitrogen-sources through the glycine metabolism. In addition, choline-O-sulphate could also be used as sulfur source [see Additional file 1]. The two added reactions represent a hypothesis that needs further experimental verification. These examples show how discrepancies between in silico predictions and physiological properties can be used to drive new discoveries, as was shown for E. coli [45].

        Growth on glucose

        P. putida KT2440, like other Pseudomonas species and rhizosymbionts, has an incomplete glycolytic pathway because of a missing 6-phosphofructokinase [70]. However, P. putida KT2440 has a complete Entner-Doudoroff pathway, which allows for the utilization of glucose and other sugars as carbon sources (Table 2). Therefore, we investigated the properties of glucose metabolism in iJN746 to validate and evaluate the reconstructed network [71]. For instance, comparison of predicted in silico growth with experimental data permits a direct assessment of the predictive potential of a reconstructed metabolic network. Subsequently, we determined the aerobic growth capability of iJN746 in Glucose-M9 minimal medium (iM9). Interestingly, iJN746 grew faster in glucose than experimental in vivo data suggested for P. putida KT2442 (Table 3, [25]). A similar difference in growth rate between in vivo and in silico measurements was reported for P. aeruginosa [64]. The difference in growth rate might be explained by an incomplete formulation of biomass function or higher energy maintenance requirements not accounted for in the current reconstruction [30, 36] or missing adaptation to glucose as primary carbon source. Another explanation could be that P. putida KT2442 converts only a part of glucose into biomass. In fact, a recent study showed that P. putida KT2442 accumulated low, extracellular concentrations of gluconate and 2-ketogluconate when grown on glucose [25]. P. putida metabolizes glucose exclusively via the Entner-Doudoroff pathway in which 6-phosphogluconate is the key intermediate. This compound is produced by three convergent pathways; the glucokinase branch, the gluconokinase branch, and the 2-ketogluconate loop (Figure 3)[70]. The latter two pathways produce gluconate and 2-ketogluconate as intermediate compounds of the glucose catabolism. iJN746 accounts for these alternate routes and corresponding transport reactions for gluconate and 2-ketogluconate.
        http://static-content.springer.com/image/art%3A10.1186%2F1752-0509-2-79/MediaObjects/12918_2008_Article_236_Fig3_HTML.jpg
        Figure 3

        Glucose metabolizing pathways present in P. putida KT2440 and its metabolic reconstruction, i JN746.

        Growth on Toluene

        Aromatic compounds such as toluene or xylene are found in polluted soil. Some Pseudomonas species are known to grow on these compounds as a sole carbon source [72], making them interesting candidates for bioremediation of contaminated areas [9, 10]. As indicated above, P. putida KT2440 can metabolize various aromatic acids, amino acids, sugars, organic acids, fatty acids, and organo-sulfur compounds (see Table 2). More specifically, P. putida KT2440 degrades many aromatic compounds into a limited number of intermediates using a few catabolic pathways that were captured in iJN746 (Figure 2). In particular, the toluene biodegradation pathway has been extensively studied in P. putida [7375] and its genetic regulation is well known [76]. In this study, we assessed the capability of iJN746 to quantitatively predict aerobic growth on toluene (Table 3). The comparison showed a much lower in silico growth rate when compared to in vivo data, 0.421 versus 0.72 (60%) (Table 3). In the following, we used different mathematical tools to elucidate reasons for this significant discrepancy.

        Reduced cost of toluene catabolism

        Linear Programming (LP) problems have two parameters, shadow price and reduced cost, which can be used to characterize the optimal solution. While shadow prices are associated with each network metabolite, reduced costs are associated with each network reaction. The reduced cost signifies the amount by which the objective function (e.g. growth rate) would increase when the flux rate through a chosen reaction was increased by a single unit [77]. Analyses of the reduced costs associated with uptake rates in the oxygen-limited toluene simulations identified the OUR as the only non-zero reduced cost value, 0.021 g biomass/gDW/h. This value corresponds to an increase of the OUR to 33 mmol oxygen/gDW/h to achieve the experimentally determined growth rate [26]. At an OUR higher than 62 mmol oxygen/gDW/h oxygen is no longer a growth-limiting factor but toluene is. Note that the upper limit of 18.5 mmol oxygen/gDW/h for the OUR was taken from measurements for E. coli corresponding to the normal oxygen diffusion rate under atmospheric oxygen conditions [78]. Mathematically, the reduced cost analysis supports the hypothesis that oxygen is the limiting factor for toluene catabolism and hence causes the reduced in silico growth rate.

        Phase Plane Analysis of toluene catabolism and oxygen uptake

        We performed a phase plane analysis to further elucidate the correlation between toluene uptake, OUR, and biomass production rate (Figure 4). We analyzed all four cases listed in Table 3 and found a direct effect of increased OUR on the toluene uptake capability and biomass production rate (Figure 4A). The experimentally observed growth rate of 0.72 μmax(h-1) [26] was achieved by TUR ranging from 6 to 11.9 mmol toluene/gDW/h and OUR higher than 33 mmol oxygen/gDW/h. Note that a higher toluene uptake rate (TUR) requires a higher OUR (Figure 4A), which indicates that the removal of intracellular oxygen was dependent on toluene availability. In fact, the three oxidative reactions involved in the conversion of toluene to 2-hydroxymuconate semialdehyde (toluene monooxygenase, benzoate 1,2-dioxygenase and catechol 2,3-dioxygenase) were found to have the higher flux rates besides the flux through the cytochrome C oxidase, an enzyme of the oxidative phosphorylation (Figure 4B).
        http://static-content.springer.com/image/art%3A10.1186%2F1752-0509-2-79/MediaObjects/12918_2008_Article_236_Fig4_HTML.jpg
        Figure 4

        (A)The phenotypic phase plane analysis showed growth rate as a function of OUR and TUR iniJN746. The growth rate is given in 1/h (color legend). The red and yellow lines represent OUR constrained to 20.93 and 33 mmol/gDW/h, respectively. (B) Diagram of oxygen producing and reducing reactions in iJN746. The flux rates are given in mmol/gDW/h and represent one possible flux state of the network in toluene minimal medium at an OUR of 40 mmol oxygen/gDW/h. The reaction abbreviations are as follows: CAT, catalase; O2tpp, oxygen periplasmic transport (oxygen uptake); ASPO6, L-aspartate oxidase; BZ12DOX, benzoate 1,2-dioxygenase; CAT23DOX, catechol 2,3-dioxygenase; CYTBO3_4pp, cytochrome oxidase bo3; DHORD, dihydoorotic acid dehydrogenase; GLYCTO1, Glycolate oxidase and XMO, toluene monooxygenase.

        In order to better understand this situation and since no detailed information about OUR was found for P. putida KT2440 under toluene-dependent growth conditions, we carried out in vivo experiments to determine the OUR of P. putida KT2440 harboring the TOL plasmid (see Methods). As expected, the OUR in toluene growing cells was higher than glucose or octanoate growing cells; 20.93 compared to 15.34 and 14.88 mmol oxygen/gDW/h, respectively (Table 3). The measured OUR uptake rate for growth in toluene did not explain the high oxygen requirement of the model, but clearly indicates the importance of oxygen uptake in toluene metabolism. Also, the measured OUR was slightly higher than the E. coli value that was used for the standard in silico simulations (20.93 vs. 18.5 mmol oxygen/gDW/h). In fact, oxygen dependent growth of toluene grown cells has been described for other P. putida strains. For example, Alagappan and Cowan reported a 10× higher oxygen-half saturation of P. putida F1 grown on toluene than other aerobic organisms [79]. Furthermore, the oxidative stress caused by toluene and other aromatic acids in the degradative process is well known [23, 80]; however, this phenomenon was found to be mainly caused by reactive oxygen species due to incomplete oxygen reduction [81], indicating an active oxygen metabolism under this growth condition. Oxygen-limiting growth conditions were also reported for P. putida when grown on octanoate [63].

        Taken together, our analysis suggests that the current P. putida metabolic network is incomplete. In fact, the current information and results suggest that the network is missing one or more reactions enabling a more oxygen-efficient catabolism of toluene and other highly reduced carbon sources (e.g. other aromatic compounds or fatty acids). This analysis represents a nice example of the broad range of applications for which iJN746 can be used to evaluate the consistency of experimental data and in silico prediction. iJN746 can serve as a platform to derive hypotheses about metabolic capabilities or missing functions in the network which can be ultimately tested in the laboratory. Hence, the metabolic reconstruction can help to increase our understanding and knowledge about this biotechnologically important organism.

        Gene essentiality analysis in iJN746

        iJN746 was used as a framework to analyze candidate essential genes in P. putida KT2440 in LB rich medium. Therefore, the network reaction(s) associated with each gene was individually "deleted" by setting the flux to 0 and optimizing for the biomass function [32]. We wished to compare the in silico essentiality predictions with experimental data to assess the predictive potential of the model. However, no large-scale, experimental gene essentiality data are available for P. putida; the information can only be found for its phylogenetic relative P. aeruginosa PAO1 and P. aeruginosa PA14 [82, 83]. A recently published comparison between the P. putida and P. aeruginosa PAO1 genomes identified 3,143 potential orthologous pairs corresponding to 60% of P. putida's total ORFs, as well as large sections of conserved gene order (synteny) [28]. Therefore, we decided to compare our in silico single gene deletion results with the 335 essential metabolic and non-metabolic genes of P. aeruginosa [82, 83]. About 12% (92) of the 746 metabolic genes present in iJN746 were predicted to be essential in iLB medium [see Additional file 2]. A total of 53% (48) of these predicted essential genes in iJN746 agreed with essential genes of P. aeruginosa [see Additional file 3]. More importantly, the 44 genes wrongly predicted as essential genes represent excellent targets for further refinement and expansion of the metabolism of iJN746 [see Additional file 4] as has been done for E. coli [45].

        False-positive predictions

        The disagreement between the experimental and computational results can reveal possible errors in the experimental data as well as in the reconstructed network. The disagreements might be caused by low experimental or sequence evidences, each of which would have hindered the inclusion of the information into the reconstruction. For example, the fabB gene was predicted to be only essential in iJN746; however, after carrying out a detailed search on Pseudomona's genomes using "The Pseudomonas Genome Database V2" http://​www.​Pseudomonas.​com/​ we found putative ORFs in the KT2440 and PA01 genome. These ORFs were annotated as alternative loci that could substitute a fabB deletion. Both, P. putida and P. aeruginosa have one copy of the fabB gene encoding for the 3-oxoacyl-(acyl-carrier-protein)synthase I (PP_4175 and PA1609, respectively). In addition, both strains have a copy of the fabF gene encoding for the 3-oxoacyl-(acyl-carrier-protein) synthase II (PP_1916 (40.92% identity with fabB-KT gene) and PA2965 (42.34% identity with fabB-PAO1 gene). Moreover, in the P. putida and P. aeruginosa genome, some ORFs were annotated putatively to encode for a 3-oxoacyl-(acyl-carrier-protein) synthase II (PP_3303 (35.94% identity) and PP_2780 (27.32% identity) in KT2440, and PA_1373 (36.17% identity) in PAO1 strain. These putative ORFs were not included in iJN746 due to the lack of supporting evidence for their metabolic function, but this analysis showed that i) PAO1 has an isozyme present in its genome, and ii) KT2440 is very likely to have at least one other ORF encoding this or a similar function. In a similar way, the discrepancy between in silico essentiality prediction and in vitro observation for msbA gene could be explained. The gene product of msbA encodes for a transporter of phosphatidylethanolamine, which is known to have a genetic redundancy in Pseudomonas sp. taking into account the Pseudomonas annotation present in "The Pseudomonas Genome Database V2". However, the supporting evidence for alternative ORFs was not strong enough to be included into iJN746.

        Finally, 37 genes were not predicted to be essential in iJN746 but they were reported as essential genes in P. aeruginosa [83] [see Additional files 4 and 3]. Of these false negatives, 13 genes encode for tRNAs synthetases which are typically included into metabolic networks [36] but are not functionally connected to the rest of the network. Hence, this disagreement was expected. Four additional false negative predictions, namely glyA (PP_0322 or PP_0671), fold (PP_1945 or PP_2265), fabZ (PP_4174 or PP_1602), and pyrH (PP_1771 or 1593), have at least one isozyme in KT2440 which were also accounted for in iJN746. For many remaining incorrectly predicted non-essential genes, the in silico deletion had a significant effect on the growth rate, reflecting their important roles in iJN746 metabolism [see Additional file 5].

        In general, many of these discrepancies suggest that metabolites enabling growth in the knock-outs might be imported from the external rich media since the exact composition of LB medium is not known [37, 38]. This observation indicates the importance of using well defined minimal media in the experimental in vivo or in vitro procedure to enable the usage of the generated data for in silico predictions and comparison.

        Gene essentiality and amino acid auxotrophy

        Jacobs et al. reported a detailed amino acid auxotroph study in P. aeruginosa PA01 using a minimal medium [82]. We carried out another single gene deletion study in glucose iM9 medium and compared the results with this PA01 study. Here, we found an absolute agreement between in vivo and in silico gene essentiality for six amino acids, namely arginine, histidine, isoleucine, valine, leucine, and tryptophan (Table 4). The presence of alternative loci in iJN746 explains partial disagreement for argA, argE, ilvA, and argJ. In fact, genetic redundancy for these genes was reported in Pseudomonas species [82]. This high correlation between in silico and in vivo data shows the utility of this approach when you take into account metabolic or anabolic reactions in a well defined minimal media. The complete lists of potential essential genes predicted in glucose iM9 medium are listed in the Additional file 6.
        Table 4

        The comparison of the in silico gene essentiality and experimental P. aeruginosa data are shown under various amino acid auxotrophic conditions.

        Amino acid

        PP gene

        gene

        Reaction

        iJN746/PA01€ (growth)

        Arginine

        PP_5185(PP_1346)

        argA†,(argJ)

        ACGS,(ORNTAC, ACGS)

        (+/-)*

         

        PP_5289

        argB

        ACGK

        (-/-)

         

        PP_3633

        argC

        AGPR

        (-/-)

         

        PP_5186,(PP_1346)

        argE†,(argJ)

        ACODA(ORNTAC, ACGS)

        (+/-)*

         

        PP_1088

        argG

        ARGSS

        (-/-)

         

        PP_0184

        argH

        ARGSL

        (-/-)

         

        PP_1346

        argJ†

        ORNTAC, ACGS

        (+/-)*

        Histidine

            
         

        PP_0292

        hisA

        PRMICIi

        (-/-)

         

        PP_0289

        hisB

        IGPDH

        (-/-)

         

        PP_0967

        hisC

        HSTPTr

        (-/-)

         

        PP_0966

        hisD

        HISTD

        (-/-)

         

        PP_5015

        hisE

        PRATPP

        (-/-)

         

        PP_0293

        hisF

        IG3PS

        (-/-)

         

        PP_0965

        hisG

        ATPPRTr

        (-/-)

         

        PP_0290

        hisH

        IG3PS

        (-/-)

         

        PP_5014

        hisI

        PRAMPC

        (-/-)

        Isoleucine-valine

            
         

        PP_3446, PP5149

        ilvA-1, ilvA-2

        SER_AL, THRD_L

        (+/-)*

         

        PP_4680

        ilvB (ilvI)£

        ACHBS, ACLS

        (-/-)

         

        PP_4678

        ilvC

        KARA1, KARA2

        (-/-)

         

        PP_5128

        ilvD

        DHAD1, DHAD2

        (-/-)

         

        PP_3511

        ilvE

        VALTA, LEUTA, ILETA

        (-/-)

         

        PP_4679

        ilvN(ilvH)£

        ACHBS, ACLS

        (-/-)

        Leucine

            
         

        PP_1025

        leuA

        IPPS

        (-/-)

         

        PP_1988

        leuB

        IPMDr

        (-/-)

         

        PP_1985

        leuC

        IPPMIa, IPPMIb

        (-/-)

         

        PP_1986

        leuD

        IPPMIa, IPPMIb

        (-/-)

        Tryptophan

            
         

        PP_0082

        trpA

        TRPS1r, TRPS3r

        (-/-)

         

        PP_0083

        trpB

        TRPS2, TRPS1r

        (-/-)

         

        PP_0422

        trpC

        IGPS

        (-/-)

         

        PP_0421

        trpD

        ANPRT

        (-/-)

         

        PP_0417

        trpE

        ANS

        (-/-)

         

        PP_1995

        trpF

        PRAI

        (-/-)

         

        PP_0420

        trpG

        ANS

        (-/-)

        The comparison of the in silico gene essentiality and experimental P. aeruginosa data are shown under various amino acid auxotrophic conditions. The in silico mutants were grown on Glucose-iM9 medium. * No auxotrophy was detected in iJN746, genetic redundancy for these genes was reported in Pseudomonas species. In P. aeruginosa mutants for orthologous genes, a significant residual growth on minimal medium was shown [82]. £ Alternative name in P. aeruginosa. € From [82].

        iJN746 as a cell factory

        In the previous section, we used the metabolic reconstruction to assess the current knowledge of P. putida's metabolism by comparing and testing in silico predictions with physiological data. However, metabolic network reconstructions can also serve as engineering and design tools [49] in addition to their use for discovery purposes [45]. Here, we investigate the poly-3-hydroxyalkanoate (PHA) production capability by the metabolic network. PHAs are a class of microbially produced polyesters that have the potential to replace conventional, petrochemically derived plastics in packaging and coating applications [63]. The biotechnological interest originates from their biodegradability and the broad range of physical properties depending on the number of carbons and side chains present in the PHA polymers [63]. These polymers are stored by many microorganisms under inorganic nutrient limited and carbon-excess growth conditions and are used as carbon- and energy sources under starvation conditions [63]. The medium-side-chain PHAs (msc-PHAs) are composed of C6 to C16 3-hydroxy fatty acids and are commonly produced by fluorescent Pseudomonas. In this way, P. putida KT2440 is an excellent candidate for msc-PHA production studies, since i) the basic msc-PHA production processes in KT2440 are well known [17, 61], ii) its genome is completely sequenced, iii) KT2440 has a well known metabolic versatility (can use a large list of carbon source as PHA precursors), iv) it is a very good host-vector biosafety system for gene cloning and expression of heterologous genes and v) this strain has been used in numerous biotechnology processes including msc-PHA production.

        iJN746 accounts for msc-PHAs ranging from C6 to C14, including two unsaturated msc-PHAs and a mixed msc-PHA polymer consisting of C8 to C12 chains. We tested the msc-PHA production capability of iJN746 from the different carbon- and energy sources listed in Table 2. All carbon sources were found to result in msc-PHA production under the chosen simulation condition (dilution rate of 0.2 hr-1). Many of these metabolites have been reported to yield in PHA production in Pseudomonas [see Additional file 7] although many studies focused on fatty acid or carbohydrate derived msc-PHAs. In general, it is assumed that carbon sources generating high levels of acetyl-CoA are good candidates for PHA production [63]. Therefore, it was not surprising to find fatty acids and carbohydrates as the best PHA precursors in iJN746 as well (Figure 5). The list of candidate (in silico) precursors includes i) L-branched-chain amino acids (L-leucine, L-isoleucine, L-Valine etc), ii) some aromatic compounds metabolized via β-ketoadipate pathway (catechol, p-coumarate, etc), and iii) other (phenylacetic acid or glycerol) (Figure 5). Interestingly, phenylacetic acid and glycerol have been reported as excellent precursors for PHA [Additional file 7]. In fact, a recent study showed that P. putida CA3 can accumulate 0.17 g of PHA per g of phenylacetate [84].
        http://static-content.springer.com/image/art%3A10.1186%2F1752-0509-2-79/MediaObjects/12918_2008_Article_236_Fig5_HTML.jpg
        Figure 5

        Maximal possible msc-PHA production rate from various carbon sources. The msc-PHA production rate is scaled per substrate carbon to facilitate the yield comparison. The simulation conditions correspond to chemostate culturing at a dilution rate of μ = 0.2 1/hr, minimal medium (iM9) supplemented with each carbon source. 'Mix' corresponds to the simultaneous production of C8:0, C6:0, C10:0, and C12:0 msc-PHA (1:1:1:1).

        Fatty acids resulted in the highest PHA production rate overall and when scaled per carbons (see Figure 5, and Additional file 7). In fact, fatty acids are converted into msc-PHAs quickly via β-oxidation [63]. Experimental studies showed that the resulting msc-PHA-monomers have the same or a smaller number of carbons as the fatty acids from which they are derived [61, 85]. In contrast, in the model, higher carbon msc-PHAs could be formed since the current model formulation does not exclude simultaneous fatty acid synthesis and β-oxidation. This situation has been experimentally demonstrated using hexanoate as a msc-PHA precursor. Huijberts et al. used inhibitors of fatty acid metabolism and demonstrated that, depending on the nature of the substrate, precursors for PHA synthesis could be derived from either beta-oxidation or fatty acid biosynthesis, and interestingly, when hexanoate was used as carbon source for msc-PHA accumulation, both routes can operate simultaneously [61]. On the other hand, the carbohydrates are converted into msc-PHA from intermediates of the fatty acid synthesis and have been shown to result primarily in C8 and C10 monomers. The model, in contrast, is able to produce the full range of msc-PHAs from carbohydrates (Figure 5). These discrepancies suggest that despite broad specificity of the Poly-(3-hydroxyalkanoate) polymerase, ranging from C6 to C16 3-hydroxy fatty acids [17], the PHA polymerizing enzyme system might have preferences for monomers with 8 or 10 carbon atoms, while larger and smaller monomers are incorporated less efficiently. This fact can also explain why, during growth on hexanoate, msc-PHA precursors are synthesized by elongation and de novo fatty acid synthesis pathway, resulting more preferably in the generation of C8 and C10 monomers [61]. Such differences in specific activity could be applied as additional constraints to the model to obtain similar results as those observed experimentally.

        Taken together, this example illustrates how iJN746 could be employed as a tool to identify new substrates (catechol, p-coumarate, isoleucine etc) for production of the different msc-PHA monomers or msc-PHA mixtures. Furthermore, computational tools such as OptKnock[86] or OptStrain[87] could help to design i) higher production strains, and/or ii) couple PHA production to growth rate. Such approaches have proven successful for other metabolic engineering designs such as lactate production in E. coli [88] or succinate production in M. succiniciproducens [89].

        Conclusion

        Here, we presented the first genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. iJN746 is a highly detailed reconstruction of the P. putida KT2440 metabolic network that captures the important biotechnological capabilities, such as biodegradation of aromatic compounds, of this paradigmatic bacterium. Moreover, iJN746 represents a comprehensive knowledge base summarizing and categorizing the information currently available for P. putida KT2440. This study evaluated the metabolic network content and showed some examples of how iJN746 could be used for biotechnological purposes. Taken together, our results underlined the value of iJN746 as a suitable tool to study of P. putida's metabolism and its biotechnical applications by the P. putida community.

        Methods

        In vivo determination of oxygen consumption and cell culture condition

        P. putida KT2440 harboring the TOL plasmid was used for in vivo determination of oxygen consumption experiments. The bacterium was grown at 30°C in M9 minimal medium [90] with octanoate (15 mM), glucose (0.3% [wt/vol]), or toluene (6 mM) as a carbon source. Liquid cultures were agitated on a gyratory shaker operated at 250 rpm. For the OUR experiment, an overnight culture of P. putida KT2440 strain grown in each carbon source was diluted until the turbidity at 600 nm (OD600) was 0.05 in fresh M9 minimal medium with the appropriate carbon source, samples were then incubated until the culture reached a turbidity at 600 nm of 0.6 for glucose or octanoate growing cells and 0.45 in toluene growing cells. Aliquots of 2 ml were taken for OUR determination; the cells were harvested by centrifugation, washed twice and re-suspended in 1 ml of fresh medium containing the appropriate carbon source using the above concentrations. The OUR was measured by monitoring the substrate-dependent oxygen consumption rate at 30°C using an oxygen electrode (DW1 Hansa-Tech Oxygen Electrode, Hansa-Tech Oxygen Instrument Limited) in 1-ml assay mixture. Cellular dry weight (CDW) was determined using previously published methods [91], using at least 3 parallel 10-ml cell suspensions that were harvested by centrifugation at 15,800 × g. The pellets were washed with 0.9% NaCl and then dried at 105°C for 24 h to a constant weight using pre-dried and weighed 2-ml Eppendorf cups.

        Network reconstruction

        The reconstruction process was done as described previously [30]. Briefly, the genome annotation of P. putida KT2440 was obtained from TIGR (http://​cmr.​tigr.​org/​tigr-scripts/​CMR/​GenomePage.​cgi?​org=​gpp, 06/27/2007) and was used as the framework of the network reconstruction. P. putida-specific primary and review literature and books were used to retrieve information about every network reaction: i) substrate specificity, ii) coenzyme specificity, iii) reaction directionality, iv) enzyme and reaction localization, and v) gene-protein-reaction (GPR) association. Relevant references were associated with every network reaction [see Additional files 7 and 8]. Public databases such as KEGG [57], PSEUDOCYC [58], and SYSTOMONAS [59] were used when no literature evidence could be found for the previous reaction characteristics. Spontaneous reactions were included into the reconstruction if i) physiological evidence suggested their presence (e.g., the presence of at least the substrate or product in the reconstruction); and ii) textbooks or KEGG [57] suggested the existence of such reactions. Every network reaction was mass- and charge balanced assuming an intracellular pH of 7.2 [38, 55]. Note that this mass- and charge balancing also included balancing the network reactions for protons (H+), water (H2O), and various co-factors (e.g., adenosine triphosphate (ATP)). No gene-associated reactions were included when no corresponding gene was annotated in P. putida's genome but physiological or experimental data supported the presence of the biochemical transformation being part of P. putida's metabolism. Finally the reversibility was determined from primary literature data for each particular enzyme/reaction, if available. This literature search resulted in a first manually-curated reconstruction specific to P. putida's metabolism based on genome annotation and available biochemical evidence. However, this list is normally incomplete and will contain network gaps that may need to be filled depending on supporting evidence. This step requires manual effort again by searching the scientific literature for supporting information. If no P. putida-specific experimental evidence could be found for a transport reaction or biochemical transformation of a metabolite, no reaction or transporter was added to the network. Finally, the network capabilities were evaluated and compared with experimental data as described in Reed et al. [30]. Detailed lists of the genes, proteins, and reactions are contained in the Additional file 8, and the definitions of all metabolites and their abbreviations are found in the Additional file 9.

        SimPheny (Genomatica Inc., San Diego, CA) software was used for the reconstruction and gap evaluation process.

        Conversion of the network reconstruction to a condition-specific model

        The reconstructed metabolic network is often represented in a tabular format, listing all network reactions and metabolites in a human-readable manner along with confidence scores and comments (see Reed et al [30] for details). The conversion into a mathematical, or computer-readable format, can be done automatically by parsing the stoichiometric coefficients from the network reaction list (e.g. using the COBRA toolbox [92]). The mathematical format is called a stoichiometric matrix, or S-matrix, where the rows correspond to the network metabolites and the columns represent the network reactions. For each reaction, the stoichiometric coefficients of the substrates are listed with a minus sign in the corresponding cell of the matrix, while the product coefficients are positive numbers, by definition. The resulting size of the S-matrix is m × n, where m is the number of metabolites and n the number of network reactions. Mathematically, the S-matrix is a linear transformation of the flux vector v = (v 1, v 2,.., v n ) to a vector of time derivatives of the concentration vector x = (x 1, x 2,.., x m ) as http://static-content.springer.com/image/art%3A10.1186%2F1752-0509-2-79/MediaObjects/12918_2008_Article_236_IEq1_HTML.gif . At steady-state, the change in concentration as a function of time is zero; hence, it follows: http://static-content.springer.com/image/art%3A10.1186%2F1752-0509-2-79/MediaObjects/12918_2008_Article_236_IEq1_HTML.gif = 0. The set of possible flux vectors v that satisfy this equality constraint might be subject to further constraints by defining v i,minv i v i,maxfor reaction i. In fact, for every irreversible network reaction i, the lower bound was defined as v i,min≥ 0 and the upper bound was defined as v i,max≥ 0.

        Exchange reactions, which supply the network with nutrients or remove secretion products from the medium, were defined for all known medium components (see Additional file 9 for details). The uptake of a substrate by the network was defined by a flux rate v i < 0 and secretion of a by-product was defined to be v i > 0 for every exchange reaction i. An exchange reaction is represented in the reaction is as follows: e.g. D-glucose exchange: Ex_glc-D: 1 glc-D →. Note that this exchange reaction is unbalanced. Exchange (uptake) reactions define the presence of media components as if one would add metabolites into an in silico flask.

        Finally, the application of constraints corresponding to different environmental conditions (e.g. minimal growth medium) or different genetic background (e.g. enzyme-deficient mutant) allow the transition from metabolic network reconstruction to condition-specific model. Note that the metabolic network reconstruction is unique to the target organism (and defined by its genome) while it can give rise to many different models by applying condition-specific constraints. All flux rates, v i , except biomass formation, are given in mmol/gDW/h.

        Biomass function

        It is generally assumed that the objective of living organisms is to divide and proliferate. Subsequently, many metabolic network reconstructions have a so-called biomass function, in which all known metabolic precursors of cellular biomass are gathered (e.g. amino acids, nucleotides, phospholipids, vitamins, cofactors, energetic requirements etc.) [3639]. Since no detailed studies about P. putida's biomass composition are available, the biomass composition from E. coli [55, 93] was used as a template for iJN746's biomass function. However, data from P. putida were added, (e.g. membrane phospholipid composition [94]), when available. The detailed calculation of the biomass composition is provided in the Additional file 10.

        in silico medium composition

        Aerobic growth was modeled in two different culture media: in silico M9 minimal medium (iM9) and in silico Luria-Bertani medium (iLB) [37]. For iM9 simulation, and according to the well described M9 minimal medium [90], the following external metabolites, CO2, Co2 +, Fe2 +, H+, H2O, Na2 +, Ni2 +, NH4, Pi and SO4 were allowed to enter and leave the network by setting the constraints on the corresponding exchange reactions (i) to v i,min≥ -106 mmol/gDW/h and to v i,max≤ 106 mmol/gDW/h. The uptake rate for each carbon source was constrained to v i,min≥ -10 mmol/gDW/h and v i,max≤ 0 mmol/gDW/h. The oxygen uptake rate (OUR) was limited to v i,min≥ -18.5 mmol/gDW/h (based on E. coli data [95]), if not noted differently. In each individual simulation, all other external metabolites were only allowed to leave the system by constraining their exchange fluxes i between v i,min≥ 0 and v i,max≥ 106 mmol/gDW/h. The iLB medium was based on the published analysis of yeast extract and tryptone provided by the corresponding manufactures, and the iLB simulations were performed according previously published methods [37].

        Phenotypic phase-plane analysis

        Phenotypic phase-plane analysis (PhPP) was carried out using SimPheny (Genomatica Inc., San Diego, CA). The underlying algorithm was described elsewhere [96, 97]. The simulation was carried out using iM9 minimal medium (as described above) and setting the bounds of toluene uptake between v i,min≥ -11.9 mmol/gDW/h (based on measurement by [26] and v i,max≤ 0 mmol/gDW/h; and of oxygen between v i,min≥ -160 mmol/gDW/h and v i,max≤ 0 mmol/gDW/h. The step size was chosen to be 35.

        Reduced Cost

        Reduced cost is a parameter of linear programming (LP) problems which is associated with each network reaction (v i ) and represents the amount by which the objective function (e.g. growth rate) could be increased when the flux rate through this reaction was increased by a single unit [77]. Reduced cost is often used to analyze the obtained optimal solution and evaluate alternate solutions from the original solution [77]. In this study, we analyzed the reduced costs associated with uptake reactions to identify candidate reactions through which an increased flux would result in a higher growth rate (under the chosen simulation condition). The growth condition was iM9 medium with toluene as carbon source. The constraints were set as described above and linear programming was employed to solve the optimization problem (maximizing growth).

        Gene essentiality and auxotrophy

        In order to determine the effect of a single gene deletion, all the reactions associated with each gene in iJN746 were individually "deleted" by setting the flux to 0 and optimizing for the biomass function [32]. A lethal deletion was defined if no positive flux value for the biomass function could be obtained for the given mutant in silico strain and medium. The simulations were performed using i) iLB rich medium for general gene essentially experiment and ii) glucose-iM9 minimal medium for auxotrophy experiments (See above). The glucose uptake rate was fixed to v i,min= v i,max= -6.3 mmol/gDW/h in the latter study. OUR was set to be v i,min≥ -18.5 mmol/gDW/h in both cases.

        msc-PHA production

        The msc-PHA production from each possible carbon source (Table 2) in iM9 medium was determined by setting the growth rate to v growth,min= v growth,max0.2 gDW/gDW/h. The lower bound of each carbon uptake reaction was set to v i,min≥ -10 mmol/gDW/h and the upper bound was set to be v i,max≤ 0 mmol/gDW/h. The lower bound of the oxygen uptake rate was set to v i,min≥ -20 mmol/gDW/h for all simulations. In iJN746, six types of msc-PHAs are defined as well as msc-PHA compounds consisting of four different carbon chains [see Figure 5 and Additional file 7]. The corresponding demand functions were used as objective functions independently for the optimization problem. The resulting msc-PHA production rates were scaled by the number of carbons of the corresponding carbon sources to facilitate a yield comparison.

        Software

        All computational simulations were performed using Matlab (The MathWorks Inc., Natick, MA) if not stated otherwise. TomLab (Tomlab Optimization Inc., San Diego, CA) was used as linear programming solver. Optimization formulations and the gene deletion studies employed the Matlab-based COBRA toolbox [92].

        Declarations

        Acknowledgements

        We thank J.R. Luque-Ortega for help in the oxygen uptake experiments. We thank M. Abrahams, M. Mo, and S. Burning for critical reading of the manuscript. JN is grateful to T. Conrad for your help during the San Diego stay and E. Díaz and M.A. Prieto for their valuable help and suggestion during the metabolic reconstruction. JN is the recipients of an I3P predoctoral Fellowship from the Consejo Superior de Investigaciones Científicas (CSIC) and JN stay in San Diego was supported by a short term I3P fellowship.

        Authors’ Affiliations

        (1)
        Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas-CSIC
        (2)
        Department of Bioengineering, University of California, San Diego
        (3)
        PhD program in Bioinformatics, University of California, San Diego

        References

        1. Clarke P, Richmond MH: Genetics and Biochemistry of Pseudomonas. New York, USA: John Wiley & Sons 1975.
        2. Clarke P: The metabolic versatility of pseudomonads. Antonie Van Leeuwenhoek 1982,48(2):105–130.View ArticlePubMed
        3. Franklin FC, Bagdasarian M, Bagdasarian MM, Timmis K: Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci USA 1981,78(12):7458–7462.View ArticlePubMedPubMed Central
        4. Bayley SA, Duggleby CJ, Worsey MJ, Williams PA, Hardy KG, Broda aP: Two modes of loss of the Tol function from Pseudomonas putida mt-2. Mol Gen Genet 1977,154(2):203–204.View ArticlePubMed
        5. Mermod N, Harayama S, Timmis K: New route to bacterial production of indigo. Bio/Technology 1986, 4:321–324.View Article
        6. Ramos J, Wasserfallen A, Rose K, Timmis K: Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkylbenzoates. Science 1987,235(4788):593–596.View ArticlePubMed
        7. Cases I, de Lorenzo V: Expression systems and physiological control of promoter activity in bacteria. Curr Opin Microbiol 1998,1(3):303–310.View ArticlePubMed
        8. Gilbert ES, Walker AW, Keasling J: A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion. Appl Microbiol Biotechnol 2003, 61:77–81.PubMedView Article
        9. Timmis KN, Steffan RJ, Unterman R: Designing microorganisms for the treatment of toxic wastes. Annu Rev Microbiol 1994, 48:525–557.View ArticlePubMed
        10. Dejonghe W, Boon N, Seghers D, Top EM, Verstraete W: Bioaugmentation of soils by increasing microbial richness: missing links. Environ Microbiol 2001,3(10):649–657.View ArticlePubMed
        11. Galán B, Díaz E, García JL: Enhancing desulphurization by engineering a flavin reductase-encoding gene cassette in recombinant biocatalysts. Environ Microbiol 2000,2(6):687–669.View ArticlePubMed
        12. Zeyer J, Lehrbach PR, Timmis KN: Use of cloned genes of Pseudomonas TOL plasmid to effect biotransformation of benzoates to cis-dihydrodiols and catechols by Escherichia coli cells. Appl Environ Microbiol 1985,50(6):1409–1413.PubMedPubMed Central
        13. Wubbolts MG, Timmis KN: Biotransformation of substituted benzoates to the corresponding cis-diols by an engineered strain of Pseudomonas oleovorans producing the TOL plasmid-specified enzyme toluate-1,2-dioxygenase. Appl Environ Microbiol 1990,56(2):569–571.PubMedPubMed Central
        14. Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B: Industrial biocatalysis today and tomorrow. Nature 2001,409(6817):258–268.View ArticlePubMed
        15. Olivera ER, Carnicero D, Jodra R, Minambres B, Garcia B, Abraham GA, Gallardo A, Roman JS, Garcia JL, Naharro G, et al.: Genetically engineered Pseudomonas: a factory of new bioplastics with broad applications. Environmental Microbiology 2001,3(10):612–618.View ArticlePubMed
        16. Ouyang SP, Luo RC, Chen SS, Liu Q, Chung A, Wu Q, Chen GQ: Production of Polyhydroxyalkanoates with High 3-Hydroxydodecanoate Monomer Content by fadB and fadA Knockout Mutant of Pseudomonas putida KT2442. Biomacromolecules 2007,8(8):2504–2511.View ArticlePubMed
        17. Huijberts GN, Eggink G, de Waard P, Huisman GW, Witholt B: Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers. Appl Environ Microbiol 1992,58(2):536–544.PubMedPubMed Central
        18. O'Sullivan DJ, O'Gara F: Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens. Microbiol Rev 1992,56(4):662–676.PubMedPubMed Central
        19. Walsh UF, Morrissey JP, O'Gara F: Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation. Curr Opin Biotechnol 2001,12(3):289–295.View ArticlePubMed
        20. Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, Martins dos Santos VAP, Fouts DE, Gill SR, Pop M, Holmes M, et al.: Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environmental Microbiology 2002,4(12):799–808.View ArticlePubMed
        21. Ramos JL: Pseudomonas. New York Kluwer: Academic/Plenum Publishers 2004.View Article
        22. Yuste L, Hervas AB, Canosa I, Tobes R, Jimenez JI, Nogales J, Perez-Perez MM, Santero E, Diaz E, Ramos J-L, et al.: Growth phase-dependent expression of the Pseudomonas putida KT2440 transcriptional machinery analysed with a genome-wide DNA microarray. Environmental Microbiology 2006,8(1):165–177.View ArticlePubMed
        23. Dominguez-Cuevas P, Gonzalez-Pastor J-E, Marques S, Ramos J-L, de Lorenzo V: Transcriptional Tradeoff between Metabolic and Stress-response Programs in Pseudomonas putida KT2440 Cells Exposed to Toluene. J Biol Chem 2006,281(17):11981–11991.View ArticlePubMed
        24. Kim Young, Sung-Ho Kun, Young Yun, Kyung-Hoon Kim, Shin Kwon, YSI Kim: Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis. PROTEOMICS 2006,6(4):1301–1318.View ArticlePubMed
        25. del Castillo T, Ramos JL, Rodriguez-Herva JJ, Fuhrer T, Sauer U, Duque E: Convergent Peripheral Pathways Catalyze Initial Glucose Catabolism in Pseudomonas putida: Genomic and Flux Analysis. J Bacteriol 2007,189(14):5142–5152.View ArticlePubMedPubMed Central
        26. del Castillo T, Ramos JL: Simultaneous Catabolite Repression between Glucose and Toluene Metabolism in Pseudomonas putida Is Channeled through Different Signaling Pathways. J Bacteriol 2007,189(18):6602–6610.View ArticlePubMedPubMed Central
        27. Jimenez JI, Minambres B, Garcia JL, Diaz E: Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environmental Microbiology 2002,4(12):824–841.View ArticlePubMed
        28. dos Santos VAPM, Heim S, Moore ERB, Stratz M, Timmis KN: Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440. Environmental Microbiology 2004,6(12):1264–1286.View ArticlePubMed
        29. Palsson BØ: In silico biotechnology. Era of reconstruction and interrogation. Curr Opin Biotechnol 2004,15(1):50–51.View ArticlePubMed
        30. Reed JL, Famili I, Thiele I, Palsson BO: Towards multidimensional genome annotation. Nat Rev Genet 2006,7(2):130–141.View ArticlePubMed
        31. Palsson BO: Two-dimensional annotation of genomes. Nat Biotechnol 2004,22(10):1218–1219.View ArticlePubMed
        32. Price ND, Reed JL, Palsson BO: Genome-scale models of microbial cells: evaluating the consequences of constraints. Nat Rev Micro 2004,2(11):886–897.View Article
        33. Becker SA, Feist AM, Mo ML, Hannum G, Palsson BO, Herrgard MJ: Quantitative Prediction of Cellular Metabolism with Constraint-based Models: The COBRA Toolbox. Nat Protoc 2007,2(3):727–738.View ArticlePubMed
        34. Price ND, Reed JL, Palsson BO: Genome-scale models of microbial cells: evaluating the consequences of constraints. Nat Rev Microbiol 2004,2(11):886–897.View ArticlePubMed
        35. Feist AM, Scholten JCM, Palsson BO, Brockman FJ, Ideker T: Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri. Mol Syst Biol 2006., 2:
        36. Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO: A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Mol Syst Biol 2007., 3:
        37. Oh Y-K, Palsson BO, Park SM, Schilling CH, Mahadevan R: Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data. J Biol Chem 2007,282(39):28791–28799.View ArticlePubMed
        38. Thiele I, Vo TD, Price ND, Palsson B: An Expanded Metabolic Reconstruction of Helicobacter pylori ( i IT341 GSM/GPR): An in silico genome-scale characterization of single and double deletion mutants. J Bacteriol 2005,187(16):5818–5830.View ArticlePubMedPubMed Central
        39. Jamshidi N, Palsson B: Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets. BMC Systems Biology 2007,1(1):26.View ArticlePubMedPubMed Central
        40. Beste DJ, Hooper T, Stewart G, Bonde B, Avignone-Rossa C, Bushell ME, Wheeler P, Klamt S, Kierzek AM, McFadden J: GSMN-TB: a web-based genome-scale network model of Mycobacterium tuberculosis metabolism. Genome Biol 2007,8(5):R89.View ArticlePubMedPubMed Central
        41. Becker SA, Palsson BO: Genome-scale reconstruction of the metabolic network in Staphylococcus aureus N315: an initial draft to the two-dimensional annotation. BMC Microbiol 2005,5(1):8.View ArticlePubMedPubMed Central
        42. Heinemann M, Kummel A, Ruinatscha R, Panke S: In silico genome-scale reconstruction and validation of the Staphylococcus aureus metabolic network. Biotechnol Bioeng 2005,92(7):850–864.View ArticlePubMed
        43. Oliveira AP, Nielsen J, Forster J: Modeling Lactococcus lactis using a genome-scale flux model. BMC Microbiol 2005, 5:39.View ArticlePubMedPubMed Central
        44. Duarte NC, Becker SA, Jamshidi N, Thiele I, Mo ML, Vo TD, Srivas R, Palsson BO: Global reconstruction of the human metabolic network based on genomic and bibliomic data. Proceedings of the National Academy of Sciences 2007,104(6):1777–1782.View Article
        45. Reed JL, Patel TR, Chen KH, Joyce AR, Applebee MK, Herring CD, Bui OT, Knight EM, Fong SS, Palsson BO: Systems approach to refining genome annotation. Proceedings of the National Academy of Sciences 2006,103(46):17480–17484.View Article
        46. Ibarra RU, Edwards JS, Palsson BO:Escherichia coli K-12 undergoes adaptive evolution to achieve in silico predicted optimal growth. Nature 2002,420(6912):186–189.View ArticlePubMed
        47. Joyce AR, Fong SS, Palsson BO: Adaptive Evolution of E. coli on Either Lactate or Glycerol Leads to Convergent, Generalist Phenotypes. International E Coli Alliance Second Annual Meeting: 2004; Banff, Alberta 2004.
        48. Fong SS, Palsson BO: Metabolic gene deletion strains of Escherichia coli evolve to computationally predicted growth phenotypes. Nature Genetics 2004,36(10):1056–1058.View ArticlePubMed
        49. Park JH, Lee KH, Kim TY, Lee SY: Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proceedings of the National Academy of Sciences 2007,104(19):7797–7802.View Article
        50. Thiele I, Price ND, Vo TD, Palsson BO: Candidate metabolic network states in human mitochondria: Impact of diabetes, ischemia, and diet. J Biol Chem 2005,280(12):11683–11695.View ArticlePubMed
        51. Ravasz E, Somera AL, Mongru DA, Oltvai ZN, Barabasi AL: Hierarchical organization of modularity in metabolic networks. Science 2002,297(5586):1551–1555.View ArticlePubMed
        52. Barabasi AL, Oltvai ZN: Network biology: understanding the cell's functional organization. Nature reviews 2004,5(2):101–113.View ArticlePubMed
        53. Almaas E, Kovacs B, Vicsek T, Oltvai ZN, Barabasi AL: Global organization of metabolic fluxes in the bacterium Escherichia coli. Nature 2004,427(6977):839–843.View ArticlePubMed
        54. Feist AM, Palsson BO: Metabolic Flux Balancing: Basic concepts, Scientific and Practical Use – 13 Years Later. Nat Biotechnol 2008,26(6):659–667.View ArticlePubMedPubMed Central
        55. Reed JL, Vo TD, Schilling CH, Palsson BO: An expanded genome-scale model of Escherichia coli K-12 ( i JR904 GSM/GPR). Genome Biology 2003,4(9):R54.51-R54.52.View Article
        56. Varma A, Palsson BO: Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110. Appl Environ Microbiol 1994,60(10):3724–3731.PubMedPubMed Central
        57. Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M: From genomics to chemical genomics: new developments in KEGG. Nucl Acids Res 2006,34(suppl_1):D354–357.View ArticlePubMedPubMed Central
        58. Romero P, Karp P: PseudoCyc, A Pathway-Genome Database for Pseudomonas aeruginosa. Journal of Molecular Microbiology and Biotechnology 2003,5(4):230–239.View ArticlePubMed
        59. Choi C, Munch R, Leupold S, Klein J, Siegel I, Thielen B, Benkert B, Kucklick M, Schobert M, Barthelmes J, et al.: SYSTOMONAS – an integrated database for systems biology analysis of Pseudomonas. Nucl Acids Res 2007,35(suppl_1):D533–537.View ArticlePubMedPubMed Central
        60. Revelles O, Wittich R-M, Ramos JL: Identification of the Initial Steps in D-Lysine Catabolism in Pseudomonas putida. J Bacteriol 2007,189(7):2787–2792.View ArticlePubMedPubMed Central
        61. Huijberts GN, de Rijk TC, de Waard P, Eggink G: 13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis. J Bacteriol 1994,176(6):1661–1666.PubMedPubMed Central
        62. Hazer B, Steinbüchel A: Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications. Appl Microbiol Biotechnol 2007,74(1):1–12.View ArticlePubMed
        63. Madison LL, Huisman GW: Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic. Microbiol Mol Biol Rev 1999,63(1):21–53.PubMedPubMed Central
        64. Oberhardt MA, Puchalka J, Fryer KE, Martins dos Santos VAP, Papin JA: Genome-Scale Metabolic Network Analysis of the Opportunistic Pathogen Pseudomonas aeruginosa PAO1. J Bacteriol 2008,190(8):2790–2803.View ArticlePubMedPubMed Central
        65. Janssen P, Goldovsky L, Kunin V, Darzentas N, Ouzounis CA: Genome coverage, literally speaking. The challenge of annotating 200 genomes with 4 million publications. EMBO Rep 2005,6(5):397–399.View ArticlePubMedPubMed Central
        66. Ryan PR, Delhaize E, Jones DL: Function and mechanism Of Organic anion exudation from plant roots. Annual Review of Plant Physiology and Plant Molecular Biology 2001,52(1):527–560.View ArticlePubMed
        67. Espinosa-Urgel M, Ramos J-L: Expression of a Pseudomonas putida Aminotransferase Involved in Lysine Catabolism Is Induced in the Rhizosphere. Appl Environ Microbiol 2001,67(11):5219–5224.View ArticlePubMedPubMed Central
        68. Stanier RY, Palleroni N, Doudoroff M: The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 1966,43(2):159–271.PubMedView Article
        69. Galvao TC, de Lorenzo V, Canovas D: Uncoupling of choline-O-sulphate utilization from osmoprotection in Pseudomonas putida. Molecular Microbiology 2006,62(6):1643–1654.View ArticlePubMed
        70. Vicente M, Canovas JL: Glucolysis in Pseudomonas putida: Physiological Role of Alternative Routes from the Analysis of Defective Mutants. J Bacteriol 1973,116(2):908–914.PubMedPubMed Central
        71. Reed JL, Famili I, Thiele I, Palsson BO: Towards multidimensional genome annotation. Nat Rev Genet 2006,7(2):130–141.View ArticlePubMed
        72. Worsey MJ, Williams PA: Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol 1975,124(1):7–13.PubMedPubMed Central
        73. Assinder SJ, PA W: The TOL plasmids: determinants of the catabolism of toluene and the xylenes. Adv Microb Physiol 1990.,31(1–69):
        74. Harayama S, Rekik M, Wubbolts M, Rose K, Leppik RA, Timmis KN: Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products. J Bacteriol 1989,171(9):5048–5055.PubMedPubMed Central
        75. Harayama S, Rekik M: The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes. Mol Gen Genet 1990,221(1):113–120.View ArticlePubMed
        76. Ramos JL, Marques S, Timmis KN: Transcriptional control of the pseudomonas tol plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annual Review of Microbiology 1997,51(1):341–373.View ArticlePubMed
        77. Ramakrishna R, Edwards JS, McCulloch A, Palsson BO: Flux-balance analysis of mitochondrial energy metabolism: consequences of systemic stoichiometric constraints. American journal of physiology 2001,280(3):R695–704.PubMed
        78. Fischer E, Zamboni N, Sauer U: High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints. Anal Biochem 2004,325(2):308–316.View ArticlePubMed
        79. Alagappan G, Cowan RM: Effect of temperature and dissolved oxygen on the growth kinetics of Pseudomonas putida F1 growing on benzene and toluen. Chemosphere 2004,54(8):1255–1265.View ArticlePubMed
        80. Denef VJ, Klappenbach JA, Patrauchan MA, Florizone C, Rodrigues JLM, Tsoi TV, Verstraete W, Eltis LD, Tiedje JM: Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400. Appl Environ Microbiol 2006,72(1):585–595.View ArticlePubMedPubMed Central
        81. Fridovich I: Superoxide radicals, superoxide dismutases and the aerobic lifestyle. Photochem Photobiol 1978,28(4–5):733–741.View ArticlePubMed
        82. Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S, Will O, Kaul R, Raymond C, Levy R, et al.: Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences 2003,100(24):14339–14344.View Article
        83. Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E, Wu G, Villanueva J, Wei T, Ausubel FM: An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proceedings of the National Academy of Sciences 2006,103(8):2833–2838.View Article
        84. Ward PG, de Roo G, O'Connor KE: Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by Pseudomonas putida CA-3. Appl Environ Microbiol 2005,71(4):2046–2052.View ArticlePubMedPubMed Central
        85. Timm A, Steinbuchel A: Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol 1990,56(11):3360–3367.PubMedPubMed Central
        86. Burgard AP, Pharkya P, Maranas CD: Optknock: a bilevel programming framework for identifying gene knockout strategies for microbial strain optimization. Biotechnol Bioeng 2003,84(6):647–657.View ArticlePubMed
        87. Pharkya P, Burgard AP, Maranas CD: OptStrain: a computational framework for redesign of microbial production systems. Genome Res 2004,14(11):2367–2376.View ArticlePubMedPubMed Central
        88. Hua Q, Joyce AR, Fong SS, Palsson BO: Metabolic analysis of adaptive evolution for in silico designed lactate-producing strains. Biotechnol Bioeng 2006.
        89. Lee SY, Lee DY, Kim TY: Systems biotechnology for strain improvement. Trends Biotechnol 2005,23(7):349–358.View ArticlePubMed
        90. Abril MA, Michan C, Timmis KN, Ramos JL: Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol 1989,171(12):6782–6790.PubMedPubMed Central
        91. Fuhrer T, Fischer E, Sauer U: Experimental Identification and Quantification of Glucose Metabolism in Seven Bacterial Species. J Bacteriol 2005,187(5):1581–1590.View ArticlePubMedPubMed Central
        92. Becker SA, Feist AM, Mo ML, Hannum G, Palsson BO, Herrgard MJ: Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox. Nat Protocols 2007,2(3):727–738.View ArticlePubMed
        93. Neidhardt FC, Ingraham JL, Schaechter M: Physiology of the bacterial cell: a molecular approach. Sunderland, Mass.: Sinauer Associates 1990.
        94. Pinkart HC, White DC: Lipids of pseudomonas. Pseudomonas. Plenum Press 111–138.
        95. Edwards JS, Ibarra RU, Palsson B: In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data. Nat Biotechnol 2001,19(2):125–130.View ArticlePubMed
        96. Schilling CH, Edwards JS, Letscher D, Palsson BO: Combining pathway analysis with flux balance analysis for the comprehensive study of metabolic systems. Biotechnol Bioeng 2000,71(4):286–306.View ArticlePubMed
        97. Edwards JS, Ibarra RU, Palsson BO:In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data. Nat Biotechnol 2001, 19:125–130.View ArticlePubMed
        98. Riley M, Abe T, Arnaud MB, Berlyn MK, Blattner FR, Chaudhuri RR, Glasner JD, Horiuchi T, Keseler IM, Kosuge T, et al.:Escherichia coli K-12: a cooperatively developed annotation snapshot-2005. Nucleic Acids Res 2006,34(1):1–9.View ArticlePubMedPubMed Central
        99. Nogales J, Canales A, Jimenez-Barbero J, Garcia JL, Diaz E: Molecular Characterization of the Gallate Dioxygenase from Pseudomonas putida KT2440: The prototype of a new subgroup of extradiol dioxygenases. J Biol Chem 2005,280(42):35382–35390.View ArticlePubMed
        100. Fan CL, Miller DL, Rodwell VW: Metabolism of Basic Amino Acids in Pseudomonas putida. Transport of lysine, ornithine, and arginine. J Biol Chem 1972,247(8):2283–2288.PubMed
        101. Vilchez S, Molina L, Ramos C, Ramos JL: Proline Catabolism by Pseudomonas putida: Cloning, Characterization, and Expression of the put Genes in the Presence of Root Exudates. J Bacteriol 2000,182(1):91–99.View ArticlePubMedPubMed Central
        102. Haywood GW, Anderson AJ, Ewing DF, Dawes EA: Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135. Appl Environ Microbiol 1990,56(11):3354–3359.PubMedPubMed Central
        103. Huisman GW, de Leeuw O, Eggink G, Witholt B: Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads. Appl Environ Microbiol 1989,55(8):1949–1954.PubMedPubMed Central

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