Multiomics Analysis Provides Insight into the Laboratory Evolution of Escherichia coli toward the Metabolic Usage of Fluorinated Indoles

Abstract

Organofluorine compounds are known to be toxic to a broad variety of living beings in different habitats, and chemical fluorination has been historically exploited by mankind for the development of therapeutic drugs or agricultural pesticides. On the other hand, several studies so far have demonstrated that, under appropriate conditions, living systems (in particular bacteria) can tolerate the presence of fluorinated molecules (e.g., amino acids analogues) within their metabolism and even repurpose them as alternative building blocks for the synthesis of cellular macromolecules such as proteins. Understanding the molecular mechanism behind these phenomena would greatly advance approaches to the biotechnological synthesis of recombinant proteins and peptide drugs. However, information about the metabolic effects of long-term exposure of living cells to fluorinated amino acids remains scarce. Hereby, we report the long-term propagation of Escherichia coli ( E. coli ) in an artificially fluorinated habitat that yielded two strains naturally adapted to live on fluorinated amino acids. In particular, we applied selective pressure to force a tryptophan (Trp)-auxotrophic strain to use either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of Trp analogues, followed by their incorporation in the cellular proteome. We found that full adaptation to both fluorinated Trp analogues requires a low number of genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity, and quality control of protein folding. These findings highlight the cellular mechanisms behind the adaptation to unnatural amino acids and provide the molecular foundation for bioengineering of novel microbial strains for synthetic biology and biotechnology.

Biochemistry of fluoroprolines: the prospect of making fluorine a bioelement

“…Efforts have been directed towards a bacterial adaptation to tryptophan-to-fluorotryptophan replacement via the engineering of the corresponding AARS [151,152]. In another approach, an adaptation of E. coli to fluorotryptophan was successfully performed using adaptive laboratory evolution (ALE) [153,154]. In ALE, the driving force for the adaptation was created by the gradual replacement of indole (tryptophan) by its fluorinated analogue, while the bacterial strain was not able to produce its own tryptophan via the native cellular machinery.…”

“…By performing experiments to achieve the proteome-wide insertion of fluoroprolines into microbial cells, we should be able to answer the question to what extent naturally evolved protein scaffolds and related cellular machineries and systems are suitable for the accommodation and integration of these isosteric building blocks. The adaptation of bacteria to fluorinated tryptophans discussed above is rather the exception, since tryptophan is a rare amino acid (only 20688 codons in the E. coli genome [77]), and hydrogen-to-fluorine replacement on the indole side-chains represent "atomic mutations", which are known to be well tolerated even in investigated isolated proteins [123,124,155] and proteomes [153,154]. Thus, it should be taken into account that the natural structural scaffolds with hydrocarbon cores have been formed and optimized through billions of years of evolution and may not be suitable to accommodate a large number of fluorine atoms [1,8].…”

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

“…Only a few rounds of selection were necessary to give rise to a 4-F-Trp-tolerating strain with mutations of rpoC, in the sigma factors sigI 15 and sigB 16 as general stress sigma factors in B. subtilis. Most recently, a similar experiment performed with Trpauxotrophic Escherichia coli revealed point mutations in the genes encoding non-essential vegetative sigma factors rpoA and rpoC, which help to reconfigure transcription under different environmental conditions, including stress 17 .…”

Meeting report of the fourth annual Tri-Service Microbiome Consortium symposium

“…Using these tools even individual diets can be assessed and predicted. More recently, these tools have been applied to research on natural products in the human gut [ 36 ] and microbial evolution to use organofluorine compounds [ 37 ].…”