A Path towards Synthetic Autotrophy in Pseudomonas putida: Engineering CO₂ fixation and light harvesting

The escalating climate crisis urgently requires innovative strategies to capture and reuse carbon dioxide. Biological CO2 upcycling is a promising route to convert greenhouse gas emissions into valuable products. Pseudomonas putida is a robust and versatile host for metabolic engineering, but it lacks the capacity of using CO2 as its main carbon source. This Thesis explores routes towards synthetic autotrophy in
P. putida by introducing the key enzymes of the Calvin-Benson-Bassham cycle. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were integrated in a selection strain designed to couple growth on ribose to CO2 assimilation. Upon introduction of the CBB shunt, growth on the selective carbon source is restored in a CO2-enriched atmosphere. This synthetic mixotrophic P. putida strain was characterized by 13C labelling analysis and its fitness was further enhanced via adaptive laboratory evolution. The final mixotrophic strain does not carry any plasmid, to avoid issues related with antibiotic usage and segregational instability. Instead, hexogenous genes were integrated in the chromosome using a novel transposon-based tool, also developed in this Thesis. Furthermore, switch from mixotrophy to full autotrophy was attempted through continous cultivation in a chemostat and targeted metabolic adjusments. In parallel, another research line explored strategies for establishing light harvesting. Autotrophic
organisms often use light to generate energy. To evaluate the portability of this phenotype, we assembled a functional proton-pumping rhodopsin in P. putida. This simple photosystem enabled light-driven ATP synthesis. Energy production was monitored in vivo by combining a genetically encoded ATP biosensor with live-cell microscopy. This Thesis contributes tools, strategies, and proof-of-concept experiments that extend the boundaries of microbial metabolism. The approach presented here transforms P. putida from a heterotrophic bacterium into a chassis capable of assimilating CO2 and harvesting light. These achievements open the way to CO2-based bioproduction and provide a framework for future development of sustainable microbial technologies.