UP Finder is an interfacial modeling tool based on the COBRA toolbox in MATLAB, which is developed by the MATLAB Graphical User Interface Development Environment (GUIDE). It is used to identify all the key gene targets for overexpression that directly related to the overproduction of a metabolite in a microorganism. The working procedure of the UP Finder is composed of following steps (Fig. 1):(1)Identification of up-regulated fluxes. The main concept is to compare the flux distributions between the wild-type and overproducing metabolic networks by calculating theoretical maximum yield of a targeted product. Thus, up-regulated fluxes and their associated pathways (termed as up-regulated pathways in this study) can be identified through this comparison.(2)Re-verification of identified pathways. Since not all the identified pathways from Step (1) are directly related to the overproduction, a re-verification is necessary to filter the low-relevant targets. For these identified pathways, their fluxes under overproducing networks were considered as the optimum fluxes (fluxopt) to achieve theoretical maximum yield of the product. The simulated product yields (Yield) constrained by each fluxopt for the wild-type network were used to evaluate the best contribution of each up-regulated pathway toward overproduction. Pathways with Yield > 0 are considered as the key targets that directly lead to the overproduction.(3)Rank of the output. The output of the UP Finder is the abbreviated reaction names of the selected key pathways in Step (2). A termed parameter, Ratio, which is the ratio of each fluxopt over their associated wild-type fluxes (fluxwt) was used for ranking the output from high to low. Because Ratio reflects the up-regulated level for each reaction, the one with the highest Ratio value suggests the highest preference when considering gene overexpression in engineering of the targeted overproduction.Fig. 1The working procedure of the UP Finder. Mutant model, the overproducing metabolic network, the model with flux distribution under the theoretical maximum yield conditions (flux distribution for reaching theoretical maximum yield of a metabolite); fluxwt, wild-type flux, which is the flux distribution of the wild-type conditions; fluxopt, optimum flux, which is the flux distribution of the theoretical maximum yield conditions; Ratio, up-regulation ratio, which is the ratio of the optimum flux to the wild-type flux of a reaction; Yield, simulated yield of the targeted product by using the optimum flux of a reaction as the constraint.Fig. 1