Harnessing the metabolic potential of Pseudomonas putida as bioproduction platform from C1 feedstocks

Carbon is the fourth most abundant element on Earth after hydrogen, oxygen and helium. Carbon-based compounds, including nucleic acids, proteins, carbohydrates and lipids, form the basis of life. In the past decades, serious concerns have been raised over anthropogenic greenhouse gas (GHG) emissions. Autotrophic carbon fixation, converting inorganic carbon into biomass components, is the main source of building blocks for life. Carbon fixation is performed through seven naturally-occurring metabolic pathways found in plants, algae and prokaryotes. Approximately 120 gigatons of carbon are cycled every year between terrestrial organisms and the atmosphere, and 90 gigatons of carbon between the oceans and the atmosphere.

The combined chemical and biological valorization of CO₂ holds the promise to contribute efficiently to reducing GHG emissions. Biological production using native autotrophs is an obvious solution, but it exhibits some limitations such as insufficient genetic tools available for their manipulation and the lack of knowledge regarding their metabolism. As an alternative, engineering synthetic one-carbon (C1) assimilation by rewiring microbial metabolism and expressing native or synthetic carbon fixation pathways in robust industrial microbes open up new avenues to turn CO₂ into a valuable resource. The soil bacterium and plant-root colonizer Pseudomonas putida has become a hefty biotechnological host due to its capacity to withstand harsh conditions and chemical stresses—highlighting its potential as a C1 assimilation host.

The work reported in this thesis harnessed the versatile metabolism of P. putida, capable of assimilating a broad range of substrates, and expanded its feedstock repertoire to accommodate formate by implementing the reductive glycine (rGly) pathway. To this end, synthetic auxotrophies were created and complemented by the functional expression of rGly pathway modules. A combination of rational and evolutionary engineering established the first reported formatotrophic P. putida strain. Subsequently, a CRISPRi-mediated gene repression approach was applied to exploit the ability of the engineered strain to accumulate value-added metabolites derived from the rGly pathway. Paired to the engineering efforts to establish a synthetic formatotrophic P. putida strain, the genetic and molecular basis of native formate, formaldehyde and methanol dissimilation was explored towards increasing the strain performance. Thus, we analyzed the transcriptome to identify genes differentially expressed in the presence of formate and methanol. Finally, native methanol and formaldehyde oxidation activities were assessed by the construction and characterization of a formate responsive transcriptional factor-based biosensor.

Overall, this thesis lays the foundation to promote synthetic C1 assimilation in P. putida and deepens our understanding of native C1 oxidation—thus bringing the formate bioeconomy one step closer to reality.