In vitro assembly of novel synthetic pathways for biohalogenation

The recent advances of synthetic biology and metabolic engineering opened the doors to an increasing number of bio-based alternatives to otherwise polluting chemical production processes. Yet, the composition of the molecules that can be produced in cell-factories remains limited to just a small number of chemical elements used for the synthesis of basic macromolecular cell components. For bio-based
processes to become a true alternative the traditional chemistry, there is a great incentive to expand the scope of elements that can be incorporated in organic building blocks. One prime example of this
untapped biochemical diversity is the bioproduction of fluorinated organic molecules (organofluorines). Organofluorines have a central role across different industrial fields, while their synthesis using biological
systems remains vastly underexplored. The fluorination pathway of Streptomyces cattleya remains the sole known biological route that enables the biosynthesis of fluorinated organic chemicals from inorganic
fluoride and a universal C1 donor S-adenosyl-L-methionine. This pathway could be the first entry point for the de novo production of organofluorines and thus a step towards the expansion of the biochemical
palette of cell factories. In this thesis, I present the characterization and optimization of bioproduction of fluorometabolites, using Pseudomonas putida as a biotechnological chassis. At the very core of this thesis is the study of each enzymatic reaction of the fluorination pathway and their optimization in purified and crude cell-extracts of P. putida. Through the selection of enzymes from diverse biological sources and their screening, an improved biofluorination pathway was assembled in a modular fashion, by mixing cell extracts from different modules of the route. This approach allowed for the enhancement of the
production of fluorinated building blocks, such as fluoroacetate (FAc), in an environment that is closer to the ones present in a living cell. By plugging-in enzymes from the reverse β-oxidation pathway, FAc can
be condensed with other carboxylic acids to generate longer and more complex fluorometabolites. To facilitate such reactions and limit the cross-talks between production routes and the innate reactions present in a crude cell-free extract, the metabolism of P. putida was engineered to reduce competitive pathways, such as the β-oxidation cycle. Overall, the modular reconstruction of an improved fluorination pathway presented here, in cell-free extracts of an engineered P. putida paves the way towards an efficient in vivo biofluorination platform. Moreover, by implementing secondary bioproduction pathways that feed from the biofluorination pathway, we developed a biological system to expand further the scope of fluorinated building blocks. Ultimately, the results of this thesis have contributed to make P. putida a biofluorination platform that is at the forefront of a new approach to the production of fluorometabolites.