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Abstract

The fixation of inorganic carbon and the conversion to organic molecules is a prerequisite for life and the foundation of the carbon cycle on Earth. Since the industrial revolution, this carbon cycle has become inbalanced and consequently the atmospheric carbon dioxide (CO2) concentration is increasing and is a major cause of global warming. On the contrary, atmospheric CO2 can also be considered as an important carbon feedstock of the future. However, human society has not yet come up with a viable solution to convert this inorganic atmospheric CO2 back into reduced carbon compounds and is still relying on natural CO2 fixation. Nature has evolved multiple solutions to reduce CO2 and incorporate it into organic molecules. The involved pathways differ in their cofactor requirements and are often limited to anoxic conditions. Many attempts have been made to improve natural carbon fixation to a more energy efficient process, but showed little success. The emerging field of synthetic biology offers an alternative approach by designing novel pathways for the fixation of CO2. Although, several such artificial pathways have been designed, none of them have been realized so far. This reveals an existing gap between the design and the realization and implementation of such a synthetic CO2 fixation pathway. In this work we designed several synthetic oxygen-tolerant CO2 fixation pathways in a bottom-up approach, by freely combining enzymes from different biological sources. The pathways were designed around an efficient central carboxylase from the family of enoyl-CoA carboxylases/reductases. Some members of this family belong to the most efficient carboxylases known so far, do not accept oxygen as a substrate and only require the ubiquitous NADPH as co-substrate. The theoretical analysis of thermodynamic and energetic properties of the designed pathways for CO2 fixation also showed that they are comparable or even more energy efficient than naturally occurring oxygen-tolerant CO2-fixing pathways. We were able to realize two of these cycles in vitro and investigated their efficiencies for the fixation of inorganic CO2 into organic molecules. We established the Crotonyl-CoA/EThylmalonyl-CoA/Hydroxybutyryl-CoA (CETCH) and HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle in vitro and their CO2 fixation efficiencies were increased in several rounds of optimization. In this process, we energized the systems by ATP- and NADPH-regeneration modules, applied the principle of metabolic proofreading to recycle undesired side products and engineered several enzymes to efficiently catalyze desired reactions. The CETCH cycle in its current version 5.4 is a reaction network of 17 enzymes originating from nine different organisms of all three domains of life. It converts CO2 into organic molecules at a rate of 5 nmol CO2 per minute and mg enzyme. In comparison, the HOPAC cycle in its current version 4.1 comprises 15 enzymes originating from eight different organisms. A stepwise incorporation of 13CO2 into the intermediates of both synthetic pathway confirmed a continuous operation for multiple rounds of conversion. During the development of the synthetic cycles for CO2 fixation, we solved a novel crystal structure of a key enzyme for both pathways, the methylsuccinyl-CoA dehydrogenase. This is a member of the well described family of flavin dependent acyl-CoA dehydrogenases. We elucidated the substrate specificity of the enzyme for (2S)-methylsuccinyl-CoA, which represents a complex substrate amongst the acyl-CoA dehydrogenase family. In summary, this study laid the foundation for the development of artificial pathways for the fixation of CO2 and narrow the gap between theoretical design of synthetic CO2 fixation pathways and their application in vivo. The CETCH and HOPAC cycle expands the solution space beyond the six naturally evolved CO2 fixation pathways by two man-made alternative that are thermodynamically more efficient than the CBB cycle of plants.