Fig. 4. In vitro biosynthesis of fluorosugars in cell-free extracts of engineered P. putida.

a Fluorination and phosphorylation steps in the canonical biofluorination pathway of S. cattleya. In the second step, 5′-fluoro-5′-deoxyadenosine (5′-FDA) is converted into 5′-fluoro-5′-deoxy-D-ribose 1-phosphate (5′-FDRP) by PNP (purine nucleoside phosphorylase). b Plasmid constructs of FluoroBricks encoding fluorinases and PNPs from Streptomyces sp. MA37 (flA1 and flB1) or S. xinghaiensis (SxflA and SxflB) under control of the P_T7 promoter, which is used in combination with the synthetic FRS-T7 RNA polymerase circuit. Phosphorylases were added with a 6× histidine tag in the N terminus as indicated. c 5′-FDA and 5′-FDRP biosynthesis in cell-free extracts of P. putida KT2440::FRS-T7RNAP bearing the FluoroBrick constructs described in (b). Data are presented as mean values and error bars correspond to standard deviations of at least three different biological replicates; An unpaired t-test statistical comparison of the data identifies differences in 5′-FDRP biosynthesis between samples, p values are shown in the figure. d ¹⁹F-NMR spectrum of fluorometabolites in cell-free extracts of engineered P. putida expressing flA1 and flB1 using the synthetic circuit, compared to the spectrum of cells bearing the empty plasmid. Source data underlying c are provided as a Source Data file.