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Background One of the European Union directives indicates that 10% of all fuels must be bio-synthesized by 2020. In this regard, biobutanolnatively produced by clostridial strainsposes as a promising alternative biofuel. One possible approach to overcome the difficulties of the industrial exploration of the native producers is the expression of more suitable pathways in robust microorganisms such as Escherichia coli. The enumeration of novel pathways is a powerful tool, allowing to identify non-obvious combinations of enzymes to produce a target compound. Results This work describes the in silico driven design of E. coli strains able to produce butanol via 2-oxoglutarate by a novel pathway. This butanol pathway was generated by a hypergraph algorithm and selected from an initial set of 105,954 different routes by successively applying different filters, such as stoichiometric feasibility, size and novelty. The implementation of this pathway involved seven catalytic steps and required the insertion of nine heterologous genes from various sources in E. coli distributed in three plasmids. Expressing butanol genes in E. coli K12 and cultivation in High-Density Medium formulation seem to favor butanol accumulation via the 2-oxoglutarate pathway. The maximum butanol titer obtained was 85±1 mg L1 by cultivating the cells in bioreactors. Conclusions In this work, we were able to successfully translate the computational analysis into in vivo applications, designing novel strains of E. coli able to produce n-butanol via an innovative pathway. Our results demonstrate that enumeration algorithms can broad the spectrum of butanol producing pathways. This validation encourages further research to other target compounds. ; This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of a Ph.D. Grant (PD/BD/52366/2013) from MIT Portugal Program and the strategic funding of UID/BIO/04469 unit. Additional support was received by COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte ...