Production of succinate with two CO₂ fixation reactions from fatty acids in Cupriavidus necator H16

Biotransformation of CO₂ into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO₂, we use fatty acids as carbon source to drive CO₂ fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16.

This work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO₂ molecules. It was verified using an isotope labeling experiment utilizing NaH¹³CO₃. This implies that 50% of the carbon atoms present in succinate are derived from CO₂, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain.

This investigation established a new method for the production of succinate by the implementation of two CO₂ fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.