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

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.

Figure 1

A. Pie chart showing the distribution of iJN746'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).

Figure 2

General depiction of the aromatic compound degradation routes present in iJN746. 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.

Figure 3

Glucose metabolizing pathways present in P. putida KT2440 and its metabolic reconstruction, iJN746.

Figure 4

(A) The phenotypic phase plane analysis showed growth rate as a function of OUR and TUR in iJN746. 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.

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).