Fluorine is almost excluded from the biotic world. Although largely ignored by nature, a life based on fluorine is both an interesting concept and an absolutely conceivable scenario. Fluorine-containing building blocks have been extensively used to investigate and modify proteins and their interactions. However, the adaptation of living organisms represents a step forward in exploring and understanding the consequences of global fluorination. Hence, a reliable platform and methodologies for the experimental evolution of novel organisms with fluorine as a bioelement were established, created by mimicking natural selection in the laboratory. In this study, adaptive laboratory evolution (ALE) was used to force Tryptophan-auxotrophic Escherichia coli bacteria to fully adapt metabolically to grow and proliferate indefinitely on 6-fluoroindole (6Fi) and 7-fluoroindole (7Fi) as substitutes for indole (Ind). During long-term cultivation, fluorinated indole precursors were supplied, in situ converted into the corresponding fluorinated amino acids, and 6-fluorotryptophan (6FTrp) and 7-fluorotryptophan (7FTrp) were incorporated into the proteome in response to the UGG codon. As consequence of the imposed selection pressure, the cells relinquished their dependence on canonical Trp and instead acquired the ability to use either of the fluorinated counterparts as an integrated part of their metabolism. In the course of this process, five independent descendant E. coli evolved that have converted their entire lifestyle to exist on these unnatural molecules. 6FTrp adaptation revealed to be superior to 7FTrp adaption. While in 6FTrp even tendencies of an indole-rejecting phenotype were observed, on the contrary, the basic adaptation to 7FTrp required extraordinary perseverance. The effect of extensive fluorination and how living organisms cope with it was investigated through comprehensive analyses of genomic, proteomic, and metabolomic levels. One major strategy found involves mitigation of the stress response system, ignoring detrimental effects caused by global fluorine integration. This study is a further step and establishes a strong foundation for further exploration of the mechanisms underlying fluorine-based life and how a former stressor (fluorinated indole) becomes a vital nutrient.
