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Existing computational tools for  de novo  metabolic 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 closer to the thermodynamic limit. Here, we introduce a two-stage computational procedure that both identifies the optimum overall stoichiometry (i.e., optStoic) and selects for (non-)native reactions (i.e., minRxn/minFlux) that maximize carbon, energy or price efficiency while satisfying thermodynamic feasibility requirements. 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 C 2+  metabolites with higher carbon efficiency.

Designing overall stoichiometric conversions and intervening metabolic reactions