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Background  Isoprenol is the basis for industrial flavor and vitamin synthesis and also a promising biofuel. Biotechnological production of isoprenol with  E. coli  is currently limited by the high toxicity of the final product. Adaptive laboratory evolution (ALE) is a promising method to address complex biological problems such as toxicity.    Results  Here we applied this method successfully to evolve  E. coli  towards higher tolerance against isoprenol, increasing growth at the half-maximal inhibitory concentration by 47%. Whole-genome re-sequencing of strains isolated from three replicate evolutions at seven time-points identified four major target genes for isoprenol tolerance:  fabF, marC, yghB,  and  rob . We could show that knock-out of  marC  and expression of mutated Rob H(48) →  frameshift  increased tolerance against isoprenol and butanol. RNA-sequencing showed that the deletion identified upstream of  yghB  correlated with a strong overexpression of the gene. The knock-out of  yghB  demonstrated that it was essential for isoprenol tolerance. The mutated Rob protein and  yghB  deletion also lead to increased vanillin tolerance.    Conclusion  Through ALE, novel targets for strain optimization in isoprenol production and also the production of other fuels, such as butanol, could be obtained. Their effectiveness could be shown through re-engineering. This paves the way for further optimization of  E. coli  for biofuel production.

Evolutionary engineering of E. coli MG1655 for tolerance against isoprenol