Escherichia coli JBEI-FM003 Genome sequencing

Engineering strains for the production of compounds of industrial interest (e.g. the biofuel precursor, limonene) using Ionic liquid (e.g EMIM Cl) treated biomass as substrate, might be limited by the toxicity of the compounds used for the pretreatment of the substrate and by the toxicity of the metabolite produced. Creating strains more tolerant to these products will be mandatory to improve the stability of the host strain and to increase the production levels. Here is a comparison of different knock-out E. coli mutants from the Keio collection (K12 BW 25113) with the wild type strain, in presence of different toxic compounds. For for the biofuel candidate, limonene and the Ionic liquid EMIM Cl, spontaneous mutants were observed in a plate based assay. These new mutants are variants of the E. coli dmsD knockout mutants and contain the plasmid pBbA8c-RFP. These mutants were isolated, and after growth on LB plate and LB liquid culture without toxic compounds, retested in the presence of limonene and EMIM Cl. The resultant strains are significantly more tolerant that the original strain, and the cause of tolerance seems stable. The “EMIM Cl” tolerant strain is not more tolerant to limonene, and vice versa, making the spontaneous mutations seem specific to the compound. These 2 mutants and the original strain were sequenced in order to identify mutations responsible for the phenotype. Sequencing these mutants will help us understand which gene(s) is (are) essential for tolerance. These knowledge will also be valuable in developing economically viable strains.