Engineering Pseudomonas putida for the targeted production of new-to-Nature fluorinated compounds

Fluorine, the most electronegative element in the periodic table, plays a pivotal role in the production of industrial compounds for modern agriculture and medicine, as well as in the manufacture of synthetic materials. The introduction of fluorine atoms into molecular structures substantially modifies their physicochemical properties. Nevertheless, this halogen is rarely found in biological systems, and approaches to integrate fluorometabolites into the biochemistry of living cells are rare. Designing cell factories for the biosynthesis of fluorinated molecules remains a significant challenge in the field of metabolic engineering, requiring not only a deep refactoring of metabolism but also the adoption of a bacterial platform that can host the harsh biochemistries involved. On this background, this thesis exploits Pseudomonas putida (P. putida) as a powerful cell factory for the production of novel fluorinated products. In order to access the rich metabolic potential of P. putida, a streamlined protocol was designed to facilitate genome engineering. Together with a comprehensive molecular toolbox, the developed method significantly reduces the overall workload needed for the genetic modification of Gram-negative bacteria and reduces the time required to introduce specific sequence alterations from up to several weeks down to six days. This toolkit was first put to the test in an applied metabolic engineering setup to establish a synthetic acetyl-coenzyme A auxotrophy in P. putida strain KT2440. A family of C2-auxotroph strains was designed to facilitate the direct selection and optimization of biochemical pathways through adaptive laboratory evolution. The functionality of the evolutionary engineering strategy was demonstrated by implementing an alternative, carbon-conserving route for sugar assimilation (i.e., the phosphoketolase shunt from bifidobacteria). Lastly, and building on all these efforts, the rich biochemical capacity of P. putida to process aromatic hydrocarbons was leveraged to produce the novel fluorinated platform chemical 2-fluoro-cis,cis-muconic acid. In this case, the genome editing toolbox was used for a profound remodeling of the native catabolism in the host to maximize bioconversion yields. Hence, the tools and platform strains developed herein enable a significant expansion of the biocatalytic landscape of P. putida through both the implementation of synthetic metabolisms and by refactoring elements of the native biochemical network.