The iJO1366 model, constructed in SimPheny, was exported as an SBML file and used to perform simulations and constraint-based analyses using the COBRA Toolbox and Tomlab CPLEX linear programming solver. The constraint-based model consists of an S matrix with 1805 rows and 2583 columns, where 1805 is the number of distinct metabolites (in all three compartments) and 2583 is the number of reactions including exchange and biomass reactions. Each of the reactions has an upper and lower bound on the flux it can carry. Reversible reactions have an upper bound of 1000 mmol gDW−1 h−1 and a lower bound of −1000 mmol gDW−1 h−1, making them practically unconstrained, while irreversible reactions have a lower bound of zero.
By default, the core biomass reaction is set as the objective to be maximized. Certain reactions are by default constrained to carry zero flux to avoid unrealistic behaviors. These reactions are CAT, DHPTDNR, DHPTDNRN, FHL (formate hydrogen lyase), SPODM, SPODMpp, SUCASPtpp, SUCFUMtpp, SUCMALtpp, and SUCTARTtpp. CAT, SPODM, and SPODMpp are hydrogen peroxide producing and consuming reactions that can carry flux in unrealistic energy generating loops. DHPTDNR and DHPTDNRN form a closed loop that can carry an arbitrarily high flux. The succinate antiporters SUCASPtpp, SUCFUMtpp, SUCMALtpp, and SUCTARTtpp can form unrealistic flux loops with other transporters for aspartate, fumarate, malate, and tartrate. The genes encoding FHL are known to be active under anaerobic conditions, but this reaction is constrained to zero to avoid unrealistic aerobic hydrogen production. The NGAM constraint is imposed by a lower bound of 3.15 mmol gDW−1 h−1 on the reaction ATPM. The exchange reactions that allow for extracellular metabolites to pass in and out of the system are defined such that a positive flux indicates flow out. All exchange reactions have a lower bound of zero except for glucose (−10 mmol gDW−1 h−1), the vitamin B12 precursor cob(I)alamin (−0.01 mmol gDW−1 h−1), and oxygen and all inorganic ions required by the biomass reaction (−1000 mmol gDW−1 h−1). The default lower bound on glucose uptake is based on typical glucose uptake rates. Because only a very small amount of B12 is required for growth, the lower bound on cob(I)alamin uptake is arbitrary and never actually constraining in practice. The iJO1366 computational model also includes drain reactions for six cytoplasmic metabolites without known consuming reactions that must be drained from the system to allow simulation of steady-state cell growth. These metabolites are p-cresol, 5′-deoxyribose, aminoacetaldehyde, S-adenosyl-4-methylthio-2-oxobutanoate, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran, and oxamate.