Succinic Acid Synthesis By Utilizing CO₂ Of Corynebacterium Acetoacidophilum

Succinic acid is widely used in chemical,food and pharmaceutical applications and is one of the most important platform chemicals for the production of various high value-added derivatives.CO₂ could be used as one of the substrates for the synthesis of butyric acid by microbial fermentation.The ability of microorganisms to fix,convert,and use CO₂ to produce chemicals is of great importance for global carbon reduction.The lactate dehydrogenase gene of Corynebacterium acetoacidophilum ATCC13870 was previously knocked out to the engineered strain C.acetoacidophilumΔldh A and a two-stage fermentation method was developed for the synthesis of succinic acid.In this study,C.acetoacidophilumΔldh A was used as the starting strain to investigate the effects of overexpression of carbon fixation enzymes and replacement of anaerobic promoters on the production of succinic acid.On the other hand,the synthesis of succinic acid by fermentation with CO₂ gas instead of carbonate,and the feasibility of linked-fermentation of ethanol and succinic acid by Saccharomyces cerevisiae and recombinant C.acetoacidophilum are further explored.Details are as follows:Firstly,the effect of enhanced carbon fixation on the synthesis of succinic acid was investigated.Four strains were constructed by heterologously overexpressing carbonic anhydrase from Synechocystis sp.PCC6803（Syn CA）,carbonic anhydrase（As CA）and phosphoenolpyruvate carboxykinase（As PCK）from Actinobacillus succinogenes,respectively.The overexpression of carbonic anhydrase in C.acetoacidophilumΔldh A had a positive effect on the utilization of CO₂ to succinic acid production.Among them,JZ002（C.aΔldh A/p JYW-5-ASca）achieved a yield of 0.72 g·g^-1,a 20.0%increase compared to the original strain,and a17.9%enhancement in the metabolic flux to the reducing tricarboxylic acid cycle.Secondly,the effect of introducing an anaerobic promoter on the synthesis of succinic acid:P_tac of C.aΔldh A/p XMJ19-pyc,the promoter of the key enzyme pyruvate carboxylase（PYC）(P_pyc)and promoter of phosphoglucoisomerase（PGI）(P_pgi)were replaced by the anaerobic promoter P_{ldh A} derived from Corynebacterium acetoacidophilum.The results showed that JZ006(C.aΔldh A/P_pyc::P_{ldh A})could reach a succinic acid yield of 0.85 g·g^-1 glucose and a concentration of 33.32 g·L^-1,which was 41.7%higher compared with the starting strain.However,the yield of succinic acid was not further improved by overexpression of Asca in the strain.Thirdly,the operation process for the synthesis of succinic acid by fermentation with CO₂gas instead of carbonate was optimized.A 3 L fermenter conversion unit connected to a CO₂gas cylinder was established,and the medium composition and conversion conditions（initial concentration of glucose,period of strain,temperature and rotational speed）were optimized.The results showed that the process with CO₂ gas inlet was simple to operate,and the concentration of succinic acid reached 34.93 g·L^-1 with a yield of 0.70 g·g^-1,which proved the feasibility of the process.Finally,a linked-fermentation process of succinic acid and ethanol was developed.Six strains of Saccharomyces cerevisiae conserved in our laboratory were screened for high ethanol production;a small-scale linked-fermentation device was designed and constructed,and the ratio of the system was adjusted;a 3-L scale linked-fermentation device was constructed,and57.92 g·L^-1 of ethanol was accumulated in 53 h,with a yield of 0.50 g·g^-1,which was close to the theoretical yield of ethanol;the yield of succinic acid was 31.55 g·L^-1,with a yield of 0.66g·g^-1.The feasibility of linked-fermentation was demonstrated,providing a new idea for green production of chemicals.