The production of succinate with more CO₂ fixation reactions facilitated by RuBisCO-based engineered Escherichia coli

Redesigning metabolic pathways to enhance the efficiency of carbon fixation during chemical biosynthesis is a promising approach for achieving cleaner and greener production of multi-carbon compounds. In this study, we established a model of cell growth in Escherichia coli that is dependent on the RuBisCO-Prk pathway by regulating its central metabolism. This rewiring ensures that growth depends on RuBisCO's carboxylation, allowing heterotrophic growth to rely on carbon fixation. This model was verified by detecting the growth curve, and it was used to screen four RuBisCO genes, of which the gene from Rhodospirillum rubrum ATCC 11170 serves as a growth advantage for E.coli. In addition, this model was applied to construct an efficient succinate biosynthetic pathway that can produce two moles of succinate from one mole of xylose and three moles of CO₂. Compared to conventional succinate biosynthesis, this strategy has a CO₂ fixation capacity that is 1.5 times greater. Furthermore, to optimize succinate production, various approaches were employed, including the optimization of key enzymes, substrate transport, and the supply of inorganic carbon. The resulting strain was capable of producing succinate at a level of 2.09 ± 0.14 g/L, which is nearly 22.4 times that of the original strain. In conclusion, this study was developed for the production of two moles of succinate by implementing three moles of carbon fixation reactions and demonstrated the feasibility of various optimization strategies in biological carbon fixation.