Designing De Novo Overall Stoichiometric Conversions and Intermediate Metabolic Reactions

Existing computational tools for the de novo pathway assembly, either based on mixed integer linear programming techniques or graph-search applications, generally only find linear pathways connecting the source to the target metabolite. The overall stoichiometry of conversion along with alternate co-reactant (or co-product) combinations is not part of the pathway design. Therefore, global carbon and energy efficiency is in essence fixed with no opportunities to identify more efficient routes for recycling carbon flux or redox equivalents closer to the thermodynamic limit. In this work, we introduce a stoichiometry and pathway design tool that first identifies an optimum overall stoichiometry by exploring exhaustively co-reactant/co-product combinations while meeting thermodynamic feasibility. Following the selection of the overall reaction, intervening reactions can be identified that link the chosen reactants and products in the desired stoichiometric ratios. Requirements on the minimality of size of the network and negativity of the free energy of change of each individual reaction step can also be imposed to help rank-order all identified designs. Implementation for recent pathway design studies identified non-intuitive designs with improved efficiencies. Specifically, multiple alternatives for non-oxidative glycolysis are generated and non-intuitive ways of co-utilizing carbon dioxide with methanol are revealed for the production of C₂₊ metabolites with higher carbon efficiency.