Elementary Flux Modes (EFMs) provide a rigorous basis to systematically characterize the steady state, cellular phenotypes, as well as metabolic network robustness and fragility. However, the number of EFMs typically grows exponentially with the size of the metabolic network, leading to excessive computational demands, and unfortunately, a large fraction of these EFMs are not biologically feasible due to system constraints. This combinatorial explosion often prevents the complete analysis of

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... ome-scale metabolic models. Traditionally, EFMs are computed by the double description method, an efficient algorithm based on matrix calculation; however, only a few constraints can be integrated into this computation. They must be monotonic with regard to the set inclusion of the supports; otherwise, they must be treated in post-processing and thus do not save computational time. We present aspefm, a hybrid computational tool based on Answer Set Programming (ASP) and Linear Programming (LP) that permits the computation of EFMs while implementing many different types of constraints. We apply our methodology to the Escherichia coli core model, which contains 226×106 EFMs. In considering transcriptional and environmental regulation, thermodynamic constraints, and resource usage considerations, the solution space is reduced to 1118 EFMs that can be computed directly with aspefm. The solution set, for E. coli growth on O2 gradients spanning fully aerobic to anaerobic, can be further reduced to four optimal EFMs using post-processing and Pareto front analysis.