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.

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

“…The biosynthetic repertoire of Pseudomonas together with implementation of heterologous pathways allows the utilisation of various substrates and synthesis of highly diverse products such as aromatics, glycolipids, and terpenoids [2][3][4]. De novo biosynthesis of fluorometabolites from mineral fluoride has recently paved a new way to fluorinated building blocks [12]. An ideal cell factory for respective biotechnological applications is composed of a chassis that exhibits inherent or engineered tolerance to the involved substrate and product, and a metabolic pathway with optimal flux between the two.…”

“…Another emerging strategy to achieve a higher and more balanced flux is to spatially organise enzymes within synthetic scaffolds [164,165] to create high local concentrations of metabolites and enzymes, with well-defined stoichiometry to support immediate conversion without diffusion of intermediates. All these strategies (Figure 3) together with an increasing understanding of tolerance traits will be instrumental for effective production applications and expanding the catalytic landscape of Pseudomonas towards novel chemistries [12,166].…”

Engineering Native and Synthetic Pathways in Pseudomonas putida for the Production of Tailored Polyhydroxyalkanoates

“…The recently demonstrated implantation of neometabolism for in vivo biofluorination in P . putida [ 159 ] will trigger further developments in this direction.…”

Recent trends in biocatalysis

“…Recently, Calero et al connected a synthetic fluorideresponsive riboswitch (FRS) to the induction of artificial metabolic pathways for the biosynthesis of fluoronucleotides and fluorosugars in engineered P. putida using inorganic fluoride as both the only fluorine source (i.e., substrate) and as the inducer of the genetic circuit. 124 The FRS post-transcriptionally (Fig. 3B, bottom) binds fluoride ions, which triggers the translation of the orthogonal T7 RNA polymerase, subsequently enabling the T7 promoter-controlled production of fluorinases and a purine nucleotide phosphorylase.…”

“…, substrate) and as the inducer of the genetic circuit. 124 The FRS post-transcriptionally ( Fig. 3B , bottom) binds fluoride ions, which triggers the translation of the orthogonal T7 RNA polymerase, subsequently enabling the T7 promoter-controlled production of fluorinases and a purine nucleotide phosphorylase.…”