Abstract:[Background] Ribulose-1,5-bisphosphate caboxylase/oxygenase (Rubisco) is a key enzyme and the rate-limiting enzyme of CO2 fixation in the Calvin cycle, and widespread in plants, algae and other autotrophic microorganisms, playing an important role in biomass synthesis and the global carbon cycle. In view of the importance and the extremely low carbon-fixation activity of Rubisco, it is of great significance to study the selection evolution of Rubisco. [Objective] This study aims to construct a selection system suitable for screening efficient Rubisco carboxylation. [Methods] The reason of the absence of selection pressure on Rubisco carboxylation in the existing system was analyzed, then the new selection system applicable for high efficiency Rubisco carboxylation was designed and constructed. The lactate-producing strain BWLac was used as the host, the growth of Rubisco expression strains with different carboxylation activities were compared under the anaerobic culture containing 5% CO2, HPLC and LCMS were used to detect total lactic acid production and the yield of lactic acid from fixed CO2 for the evaluation of new constructed selection system. [Results] The design principle was considered to base on the pull-down force generated by the terminal metabolite lactic acid and the balance of the residual NADH generated by glycerol metabolism. These two parts could enhance the metabolic flux of the carbon-fixing branch of Prk and Rubisco, which could strengthen the inhibition of the toxicity of RuBP, coupling cell growth effectively with Rubisco activity to construct a high-throughput screening approach. In the newly constructed selection system, the Rubisco inactivated mutant BWLac/197 could not grow, while the growth of Rubisco and Prk double inactivated mutant BWLac/197-2021 was not affected with the colony size was about 1.58 cm. RBC1 and 7002, which carboxylation activities were detected to be more than 2 times different, the cell growth was inhibited to varying degrees under the screening conditions, and the colony size was 1.06 cm and 0.65 cm, respectively. The lactic acid production and glycerol consumption were also tested to evaluate the effectiveness of the selection system. RBC1 consumed 1.39 g/L of glycerol, produced 2.82 g/L of lactic acid, and labeled lactic acid content of 18.05 μmol/L, which was 1.3?1.6 times higher than the corresponding test result of 7002. The detection results were consistent with the design principle of the selection system, the higher the lactic acid production, the more glycerol consumption, the higher the Rubisco carboxylation, the better the cells grow. [Conclusion] An efficient selection system for Rubisco carboxylation was successfully designed and constructed to provide an effective high-throughput screening method for the evolution or exploration of Rubisco with higher carboxylation activity.
