Engineering Pseudomonas putida KT2440 towards in vivo implementation of a synthetic CO2 fixation pathway

Abstract

As atmospheric CO2 levels continue to rise, threatening global climate stability, carbon fixation cycles offer a promising dual solution: they not only help reduce greenhouse gas concentrations but also provide a sustainable pathway to produce valuable molecules directly from air, turning an environmental challenge into a resource. Synthetic biology approaches enable the design of synthetic CO2 fixation pathways never exhibited in nature. One example is the crotonyl-coenzyme A (CoA)/ethylmalonyl- CoA/hydroxybutyryl-CoA (CETCH) cycle, a synthetic CO2 fixation pathway designed to operate faster and more efficient than the naturally evolved Calvin-Benson-Bassham cycle. The functionality of the CETCH cycle has been extensively validated through in vitro approaches. To advance toward biological implementation, we engineered Pseudomonas putida KT2440—a metabolically versatile, NADPH-rich chassis—to host modular CETCH components. To achieve this, we divided the CETCH pathway into four functional modules, expressing two on plasmids to test integration into native metabolism. Growth experiments revealed that strains expressing CETCH components exhibited enhanced growth on crotonate compared to control strains, particularly when methylmalonyl-CoA mutase activity was included. Through genetic engineering, we identified PP_3553 as a previously uncharacterized crotonyl-CoA ligase essential for crotonate metabolism in P. putida. To optimize flux through the CETCH pathway, we generated knockout strains (ΔFadB and ΔPP_3553) that eliminated native crotonate metabolism pathways. Metabolomics analyses showed limited carbon flux through the complete CETCH cycle, suggesting potential metabolic bottlenecks. Using auxotrophic sensor strains, we validated partial CETCH functionality and successfully evolved a strain with improved growth characteristics. This work demonstrates partial implementation of a synthetic carbon fixation pathway in P. putida and provides a foundation for future engineering efforts to develop more efficient biological carbon capture systems.