Synthetic Fluorine Metabolism: Expanding the Boundaries of Microbial Biochemistry for Organofluorine Biosynthesis

Research output: Book/ReportPh.D. thesis


Fluorine (F) is the 13th most abundant element on the Earth’s crust, just ahead of Carbon. Despite its pervasiveness, the presence of F in the biosphere is negligible, and only few tens of organofluorines are known today. Organofluorines are compounds of extreme value, widely used in many fields such as pharmaceuticals, agriculture and materials. Our society benefits from several fluorinated products, and none of them has a biological origin. Thus, F holds a high importance for materials development and biotechnological solutions. The scarcity of natural organofluorines results in a narrow biochemical spectrum and the consequent scarceness of specialized biocatalysts. Much of the biochemistry of halogens (X) involve the oxidation of halide ions (X–) to halonium ions (X+) or halide radicals (X·). Due to the extreme electronegativity of the F atom, such an oxidation approach is unfeasible. To date, only one family of enzymes have recognized to catalyse the formation of a C–F bond: fluorinases. The first enzyme of this kind was discovered in the soil bacterium Streptomyces cattleya, encoded in the biosynthetic gene cluster of the fluorination pathway responsible for the synthesis of virtually all know natural organofluorines. Because of its uniqueness, such pathway is the cornerstone for the construction of cell factories for de novo biosynthesis of organofluorines. Since its discovery, the fluorination pathway has been identified in other members of the Actinomycetes phylum and new enzyme variants are becoming accessible. Furthermore, due to the small steric hindrance of the F atom, which has a slightly smaller atomic radius than Hydrogen, is not uncommon for enzymes to promiscuously accept fluorinated substrates. This property can lead to the development of new–to–nature biochemistries in microbial factories and new manufacturing technologies for organofluorines.
In this work, I have investigated how foreign fluorinated metabolites can alter the metabolism of the Gram–negative bacterium Escherichia coli and how they are naturally assimilated into the metabolic network. Synthetic routes for assimilation and de novo synthesis of different organofluorines were then introduced in two well–known bacterial workhorses: E. coli and the non–pathogenic soil bacterium Pseudomonas putida. Finally, I investigated how enzymes promiscuity can be exploited to catalyse novel reactions for the biosynthesis of the non–canonical amino acid 4–fluoro–L–threonine. From a broad perspective, this study characterizes different aspects of natural and synthetic F–metabolism, combining observations of bacterial biochemistry and exploring the biotechnological potential of expanding a ‘fluorinated’ metabolism with synthetic biology tools. The findings open the path for further development of robust cell factories for biobased production of difficult–to–produce organofluorines, as well as the biosynthesis of new–to–nature compounds of commercial interest.
Original languageEnglish
PublisherTechnical University of Denmark
Number of pages185
Publication statusPublished - 2023


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