Team:Valencia UPV/Modeling/fba

COBRA

The COnstraints Based Reconstruction and Analysis (COBRA) approach to systems biology accepts the fact that we do not possess sufficiently detailed parameter data to precisely model, in the biophysical sense, an organism or plant at the genome scale [1]. The COBRA Toolbox [2] is a freely available Matlab toolbox can be used to perform a variety of COBRA methods, including many FBA-based methods. Models for the COBRA Toolbox are saved in the Systems Biology Markup Language (SBML).

AraGEM Model

AraGEM is the genome-scale metabolic network model covering primary metabolism for a compartmentalized plant cell based on the Arabidopsis (Arabidopsis thaliana) genome. AraGEM is a comprehensive literature-based, that accounts for the functions of 1,419 unique open reading frames, 1,748 metabolites, 5,253 gene-enzyme reaction-association entries, and 1,567 unique reactions compartmentalized into the cytoplasm, mitochondrion, plastid, peroxisome, and vacuole.

Using efficient resource utilization as the optimality criterion, AraGEM predicted the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. Hence, we have added to AraGEM in silico the reactions from the network of production pheromone. The optimality criterion finds to maximize the pheromone flux, which is produced by the plant, and it is released into the air to confuse the moths.

AraGEM is a viable framework for in silico functional analysis and can be used to derive new, nontrivial hypotheses for exploring plant metabolism. AraGEM is the genome-scale metabolic network model that better approximates the metabolism model of N. benthamiana.

Pheromone pathway location

Figure 1. Palmitic acid is a common fatty acid in plants and animals. This metabolite is the precursor of our pheromone.

Palmitic acid (16:0) is the most common fatty acid (saturated) found in animals, plants and microorganisms. Coenzyme A (CoA) also presents in living organisms, is bound to Palmitic acid for synthesis and oxidates several fatty acids.

These 2 elements reacts to form the first metabolite of our pathway 16:CoA, which is generated by the endoplastic reticulum (plastid membrane).

AraGEM model is compounded by 1737 metabolites and 1601 reactions. These elements are organized in the stoichiometric matrix S[1737x1601] to use them in the FBA optimization take into account some constraints.

Our efforts were aimed at the optimization of Palmitic Acid or Hexadecanoic32 Acid_acc (16:0), which is the precursor of our pathway. 16:0 is located in the cytosol and the reaction takes place using one influx V1 and two effluxes V2, V3. The efflux V2 was incorporated as an exchange reaction in order to generate branch in the original pathway of 16:0 metabolism. As our metabolic pathway is linear starting from 16:0, this new added branch represents the hole synthetic pathway of our pheromone.

Figure 2. In AraGEM, palmitic acid is also called Hexadecanoic32 Acid_acc. The reaction added ExZ11 produces the efflux V3 known as palmitic acidexchange.

Defining scenarios

AraGEM is a generic plant cell model capable of representing both photosynthetic and nonphotosynthetic cell types [1]. In order to reproduce the classical physiological scenarios of plant cell metabolism, we explored two photosynthetic scenarios: i) Photosynthesis and ii) Photorespiration. The difference comes from the carboxylation reaction of Rubisco. This enzyme in the presence of oxygen (O2) reduces the energy efficiency of photosynthetic output by 25% in C3 plants.

Figure 3. Rubisco enzyme action defines two scenarios in plant metabolism: Photosynthesis and Photorespiration. During Photosynthesis, the carboxylation-oxygenation ratio is +1:0 and there is no oxygenation of ribulose 1.5-bisP. Moreover, the presence of oxygen and rubisco also catalyzes an oxygen reaction, photosynthesis efficiency is reduced and the carboxylation-oxygenation ratio is +3:1.

Model assumptions

In plants, the most genome-scale metabolic network reconstructions minimize the photons consumption (Ex16) while growth rate or biomass (BIO_L) rate is kept fixed. This is true if we mainly seek the survival of the plant, and photons consumption will be the optimization objective.

In our case, the principal objective is maximizing the pheromone production, in other words, optimizing the efflux V3 from the exchange reaction ExZ11. In addition, we must introduce the low (lb) and up (ub) bound for each reaction. In AraGEM, the bounds for flux reactions Ex16, BIO_L and ExZ11 were fitted according to Table 1.

Table 1. Upper and lower bounds in AraGEM.

Optimal pheromone production using FBA

Table 2. FBA results in each scenario.

The optimum flux distribution is here defined as the flux distribution that maximizes palmitic acid flux, for a fixed rate of biomass synthesis and free photon uptake.

The metabolic contrast between photosynthesis and photorespiration is illustrated in Figure 4. Photosynthesis (Figure 4A and 4B) shows an increase of the palmitic acidexchange flux, when the photons uptake are unconstraint and takes values until 1000 (default maximum flux COBRA).

Even when the photons flux freely increase, the most palmitic acid is used by the biomass or plant growth. Therefore, the new palmitic acid_{exchange} flux only becomes 5.33 nM/seg. However, this level also depends of the high growth rate fixed from the literature.

Figure 4. Optimization during photosynthesis with two different objectives fluxes A) minimizing photons uptake and B) maximizing palmitic acidexchange. During photorespiration, metabolism products decrease, such as C) photons uptake and D) palmitic acidexchange.

During photorespiration, we used FBA to optimize and analyze both palmitic acidexchange and photons uptake. Figure 4C and 4D shows an imperceptible loss (2%) in the palmitic acidexchange flux. The new flux is 5.24 nM/seg and its behavior is similar to the photosynthesis scenario.

We included a supplementary analysis produced by FBA optimization called Gene knockout. COBRA toolbox knock outs one by one gene of the AraGEM model to optimize the desired objective flux. The Figure 5 shows the maximum palmitic acidexchange flux obtained after gene knockout of each gene During photosynthesis or photorespiration, the optimal palmitic acidexchange flux presents a decrease when around 33 from 1404 gene were knocked out.

Figure 5. Palmitic acidexchange flux after gene knockout. From 1404 gene in AraGEM model, 33 (pink lines) gene show an influence over the optimal Palmitic acidexchange flux.

References