Recent advances in computational biology led to emergence of databases with whole-genome metabolic networks. They describe the flow of metabolites within a particular organism. Usage of constraint-based method allows to make quantitative predictions about the phenotype while eliminating many of the complex parameters. Of particular interest to our project is flux balance analysis (FBA), which allows to predict the optimal steady-state biomass flux, which is often correlated with cell growth rate[1] and is the most likely observed phenotype. One major advantage of FBA is that it does not require knowledge of enzymatic parameters. The mathematics of FBA is elucidated in this figure[2] One of the primary questions for the project is the following: will the synthetic P. denitrificans strain grow better than the unmodified one in presence of ammonia? To answer this question, we performed comparative analysis of the two Paracoccus strains using FBA on a whole-genome metabolic model. The analysis pipeline involved a slew of open-source computational tools, which we will describe below.
First, we reconstructed a metabolic model of Paracoccus denitrificans strain DSM 413 on a complete medium using ModelSEED[3]. A complete medium is such that any nutrient, including ammonia, is available for uptake. Thus, the set of reactions included in the model is the biggest of all possible sets. Although the largest, this set is incomplete. In the next step, the model was gapfilled with all the reactions necessary for measurable cell growth. The nature of our project dictates that we must be able to manually include several reactions, metabolites and genes (e.g. oxygenation of ammonia by the AMO enzyme complex) into the model. Such functionality is not available in ModelSEED. To implement this, we turned to COBRApy: Constraint-Based Reconstruction and Analysis[4] package written in Python. COBRApy does not natively work with ModelSEED models. To overcome this, we used Mackinac package[5] to convert the ModelSEED model into COBRApy. Using COBRApy, we can then add the new ingredients into the model and perform FBA to compare the biomass fluxes, and hence the growth rates, of the two Paracoccus strains. The script is available here. First, the FBA was run on the gapfilled unmodified model, which initially contained 1550 reactions and 1556 metabolites. The optimal biomass flux was found to be \( 224.3248 \frac{\text{mmol metabolite}}{\text{g dry weight}\cdot\text{h}} \). Next, we add the reactions. Below is the list of all reactions added to the model. \(\ce{Q}\) and \(\ce{QH_2}\) represent ubiquinone and ubiquinol, respectively. \( \text{UqO} \) is the ubiquinone oxidoreductase enzyme which catalyzes the last reaction.
\[ \ce{NH_3 + QH_2 + O_2 ->[\text{AMO}] H_2O + Q + NH_2OH} \]
\[ \ce{NH_3 + NAD + H_2O ->[\text{AMO}] 2H^+ + NADH + NH_2OH} \]
\[ \ce{NH_2OH + O_2 ->[\text{HAO}] NO_2^- + H^+ + H_2O} \]
\[ \ce{NH_2OH + 2Q + H_2O ->[\text{HAO}] NO_2^- + 2QH_2} \]
\[ \ce{QH_2 ->[\text{UqO}] 2H^+ + Q} \]
With the new model containing 1555 reactions and 1559 metabolites (hydroxylamine, Q and QH2 added), the optimal biomass flux of the modified model is \( \boxed{228.6980 \frac{\text{mmol metabolite}}{\text{g dry weight}\cdot\text{h}}} \).
Results and Discussion
Because the modified biomass flux is greater than the original flux and the difference (2%) is significant, we can conclude that the cell makes use of the inserted nitrification pathway in order to enhance its metabolism. Thus, our device confers fitness advantage. One possible implication of that is it is possible that our synthetic strain will out-compete the native strain inside the sludge, thus eliminating the need to artificially sustain the new culture. As was stated earlier, the expected cell phenotype is the one with the largest biomass flux. This is only true under the assumption that the cell lives in an ideal or near-ideal environment. Several studies have shown that under different growth environments, cells sometimes exhibit non-optimal yield metabolism. We do not know whether wastewater causes a similar shift in metabolism of Paracoccus denitrificans. However, seeing as it is able to thrive in such environment gives enough reason to believe that the growth rate will not lose correlation with biomass flux.
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