Fig. 2: In vitro biofluorination in cell-free extracts of engineered P. putida.

a Biofluorination reaction, indicating the transformation of SAM (S-adenosyl-L-methionine) and F⁻ into 5′-FDA (5′-fluoro-5′-deoxyadenosine) by the fluorinase enzyme. b 3D structure of the trimer form of fluorinase (PDB ID 1RQP); SAM bound to the enzyme is represented as yellow spheres. c Mechanism of action of the fluorinase enzyme. Functional groups that interact with F⁻ (magenta) are indicated in blue. d Scheme of the synthetic circuit designed for fluoride-triggered biofluorination. The T7 RNA polymerase gene (dark green) is integrated in the genome; upon exposure to F⁻ (pink squares), the P_T7 → flA1 module (light blue, borne by plasmid pFB·1F1) is expressed and the FlA1 fluorinase (light blue circles) is produced, catalyzing the conversion of SAM (green pentagon) and F⁻ into 5′-FDA. FluoroBrick 1, containing an RBS (yellow circle) and flA1, is boxed with dashed green lines. e FluoroBricks tested for 5′-FDA biosynthesis. Genes encoding fluorinases from Streptomyces sp. MA37 (flA1) and S. xinghaiensis (SxflA) were combined with the XylS/Pm expression system or the FRS-T7 RNA polymerase circuit described in (d) by modular assembly. f 5′-FDA biosynthesis in cell-free extracts of P. putida KT2440 bearing the FluoroBrick constructs described in (e). Data are presented as mean values and error bars correspond to standard deviations of at least three different biological replicates. An unpaired t-test analysis was used to compare 5′-FDA biosynthesis across experiments, with one asterisk indicating a p value = 0.0410 and two asterisks identifying a p value = 0.0121. g ¹⁹F-NMR spectra of the cell-free extracts. Key chemical shifts corresponding to 5′-FDA are indicated. Source data underlying Fig. 2f are provided as a Source Data file.