Refactoring native metabolism in Pseudomonas putida towards production and incorporation of novel fluorinated building blocks

Breaking boundaries within chemistry is at it’s core the way we, as a society, reach further heights in synthesis a novel compounds and materials to better the lives of the people we live with. Development of polychlorotrifluorethylene in the late 1930s birthed Teflon, tetrafluoroethylene, which was extensively used during Manhattan project for uranium enrichment during World War II. Similarly, the chemical bounds of bacterial metabolism represent a crucial barrier to expanding our utilization of cell factories, factories which have been gaining speed and popularity as alternative for traditional chemistry in the past two decades. In order to break these bounds, researchers need to take advantage of the promiscuity of enzymes and the versatility of chemical synthesis.

In this thesis I first elucidate the promiscuous nature of sulfonate metabolism in Pseudomonas putida and develop tools for its adaptation. By using the knowledge generated from Escherichia coli, I choose disruption targets which allow for maximum elucidation of this underexplored metabolism. Further omics characterization shed light on the regulational mechanisms of total sulfur metabolism. Next, I explore upgrades to the versatile and cheap Chi.Bio reactor system for automated adaptive laboratory evolution (ALE) with P. putida in mind. At the core of this thesis, I synthesize the sulfur metabolism exploration with advances to automated ALE in an attempt to build a platform strain of P. putida capable of breaking the bounds of conventional biochemistry. The attractive promiscuous nature of sulfonate metabolism is balanced by the low demand for sulfur molecules. Then, I attempt to build a biosynthetic shunt for medium chain fatty acids, with PFAS entry as guiding principal to secondary metabolism in mind. The core of this thesis is an attempt at the expansion of the metabolic landscape in Pseudomonas putida with the concomitant elucidation of underexplored metabolisms. In doing so, we found that, for the most part, sulfur metabolism follows a similar model as E. coli and very few modifications are required to enable fatty acid shunting.

Overall, the results in this thesis set the foundation for establishing a platform for novel chemistry discovery. Additionally, they provide an avenue for bioremediation of PFAS, the worlds pre-eminent environmental pollutant. If key metabolic hurdles are overcome, the strains and work established here-in would set the stage for production of molecules which, at their core, are New-to-Nature.