Application of OptORF to an example metabolic and regulatory network. (A) In this example network, substrate S is utilized to produce biomass B and by-products P1 or P2. Here, the engineering objective is the production of P1 and the cellular objective is the production of B. Since reaction R5 produces more B than reaction R2, P2 is the preferred by-product and P1 production is uncoupled to biomass production (no P1 is produced when biomass is maximized). (B) Reactions in the network and their GPR associations. (C) Transcriptional regulation of the metabolic genes and transcription factor in the network. (D) Metabolic engineering strategies for the production of P1 identified by OptORF, where P1 is now produced when biomass production is maximized in the altered networks. (E) A schematic view of the OptORF solutions. The metabolic flux distributions for wildtype and mutants are shown to describe how the optimal flux profiles (where production of biomass, B, is maximized) in the integrated network changes by the metabolic and/or regulatory perturbations presented in (D).