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A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida
Abstract
Fluorine is a key element in the synthesis of molecules broadly used in medicine, agriculture and materials. Addition of fluorine to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to integrate fluorometabolites into the biochemistry of living cells are scarce. In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida . By harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, biochemical reactions needed for in vivo biofluorination are wired to the presence of fluoride (i.e. circumventing the need of feeding expensive additives). Biosynthesis of fluoronucleotides and fluorosugars in engineered P . putida is demonstrated with mineral fluoride both as only fluorine source (i.e. substrate of the pathway) and as inducer of the synthetic circuit. This approach expands the chemical landscape of cell factories by providing alternative biosynthetic strategies towards fluorinated building-blocks.
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There is a parallel interest in using Pseudomonas as a vector for the biosynthesis of new fluorinated compounds, some of which may serve as substitutes for synthetic fluorinated chemicals.
“There is a parallel interest in using Pseudomonas as a vector for the biosynthesis of new fluorinated compounds (9, 46, 59), some of which may serve as substitutes for synthetic fluorinated chemicals.”
From Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion (2024) · cites this paper as [46]
The intracellular F− concentration in P. putida strains carrying pS441·FRSv1 is correlated with the msfGFP fluorescence output.
“The intracellular F− concentration in these strains was correlated to the msfGFP fluorescence output.”
From Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride (2022) · cites this paper as Calero et al., 2020
The FRS can be indirectly used as a means to assess F− concentrations owing to the conformation change in the secondary mRNA structure in the presence of the anion.
“The FRS can be indirectly used as a means to assess F− concentrations owing to the conformation change in the secondary mRNA structure in the presence of the anion.”
From Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride (2022) · cites this paper as Calero et al., 2020
The conformational transition of the FRS exposes a buried ribosome binding site, leading to the translation of the reporter msfGFP.
“This conformational transition exposes a (buried) ribosome binding site, leading to the translation of the reporter (i.e. msfGFP, a monomeric superfolder GFP).”
From Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride (2022) · cites this paper as Calero et al., 2020
The effects of F− salts on the physiology of Pseudomonas putida remained unstudied prior to this work.
“yet the effects of these salts on the bacterial physiology remain unstudied”
From Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride (2022) · cites this paper as Calero et al., 2020
CrcB functions as a passive ion channel and is considered a primary defense against fluoride toxicity in many bacteria, including Escherichia coli, Bacillus subtilis, and Pseudomonas species.
“CrcB functions as a passive ion channel and is considered a primary defense mechanism against fluoride toxicity in many bacteria, including Escherichia coli, Bacillus subtilis, and Pseudomonas species (5–7).”
From Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter (2026) · cites this paper as [7]
The CrcB export protein, also known as Fluc, exports intracellular fluoride ions.
“Previous studies have highlighted the role of the CrcB export protein (also known as Fluc) in exporting intracellular fluoride ions (7).”
From Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter (2026) · cites this paper as [7]
The intracellular F− concentration in the strains carrying pS441·FRSv1 correlated with msfGFP fluorescence.
“The intracellular F− concentration in these strains correlated with msfGFP fluorescence.”
From Adaptive evolution of Pseudomonas putida in the presence of fluoride exposes novel functions of a benzoate transporter (2026) · cites this paper as [7]
A knockout of the crcB gene, encoding a membrane protein that exports fluoride, makes P. putida KT2440 and other bacteria much more sensitive to fluoride.
“Previous studies had also shown that a knockout of the crcB gene, encoding a membrane protein that exports fluoride, makes P. putida KT2440 (46) and other bacteria (13) much more sensitive to fluoride.”
From Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion (2024) · cites this paper as [46]
Fluoride powerfully inhibits metabolism.
“Both efforts are undermined by the substantial toxicity of fluoride, an anion that powerfully inhibits metabolism.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [22]
Fluoride is highly toxic to the bacteria carrying out biosynthetic or biodegradation reactions.
“Though NaF is much safer for humans than HF and fluorine gas, fluoride is highly toxic to the bacteria carrying out biosynthetic or biodegradation reactions21,22.”
From The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis (2024) · cites this paper as [22]
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Stockbridge, R. B., & Wackett, L. P. (2024) · Nature Communications · doi:10.1038/s41467-024-49018-1 · Cited by 80
Calero, P., Gurdo, N., & Nikel, P. I. (2022) · Environmental Microbiology · doi:10.1111/1462-2920.16110 · Cited by 56
Self-citationDodge, A. G., Thoma, C. J., O’Connor, M. R., & Wackett, L. P. (2024) · mBio · doi:10.1128/mbio.02785-23 · Cited by 23
Ets, L., Ilves, H., Juhe, L., Ilmjärv, T., Puiggené, Ò., Nikel, P. I., & Kivisaar, M. (2026) · Journal of Bacteriology · doi:10.1128/jb.00479-25 · Cited by 1
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Metadata
- Authors
- Calero, P., Volke, D. C., Lowe, P. T., Gotfredsen, C. H., O’Hagan, D., & Nikel, P. I.
- ORCID
- Nikel, Pablo I.
- Venue
- Nature Communications
- Published
- 2020-10-07
- Volume
- 11
- Issue
- 1
- ISSN
- 2041-1723
- PMC
- PMC7541441
- Cited by
- 97
- References
- 1
- Language
- en
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