Study On Succinic Acid Production By Corynebacterium Glutamicum

Corynebacterium glutamicum is a classic organism in amino acids fermentative production. Exploring metabolic engineering C. glutamicum for succinic acid production has been significant in both development new processes of bio-succinic acid production and uses of C. glutamicum. In this work, the metabolic pathway of succinic acid production by C. glutamicum and strain improvement, including traditional mutagenesis and gene knock-out technology were studied.First, C. acetoacidophilum ATCC 13870 were tested to accumulate highest concentration of organic acid among fourteen L-glutamate acid-producing strains preserved in our lab. Further study showed that the yield of organic acid increased with its cell density. And the optimal pH vale for succinic acid and lactic acid generated were 7.5 and 8.0, respectively, at which the concentrations of succinic acid and lactic acid reached 22.5 g/L and 60.0 g/L, correspondingly. Bicarbonate was the key factor influenced organic acid synthesis. The metabolic flux analysis of ATCC 13870 at sodium bicarbonate concentration of 40 and 400 mmol/L indicated that PEP node was evidently influenced by sodium bicarbonate concentration but the influence of PYR node is less. Suggesting to block its metabolic bypass to lactic acid might be leading carbon flow to succinic acid more.Second, C.acetoacidophilum ATCC 13870 were mutated by acriflavine-UV treatment. A strainⅡ-169 with a yield of 23.8 g/L was obtained, which yielded 14.4% more succinic acid and 12.5% less lactic acid than ATCC 13870. But lactic acid is still high (52.5 g/L).The lactate dehydrogenase gene (ldh) of ATCC13870 was knockouted by the double-crossover chromosome replacement with sacB gene. The upstream or downstream region of the ldh gene was amplified and then performed by the second round of overlap-extension PCR. The fused PCR fragments were ligated with pK19mobsacB to composed pK19mobsacB-Δldh and electroporated into C.acetoacidophilum ATCC 13870. A ldh-deleted mutant C.acetoacidophilumΔldh was obtained by two rounds of selection with Kanamycin resistance and sucrose selection pressure identified by enzymatic activity measurements. The successful knockout of ldh from the chromosome was confirmed by PCR and enzymatic activity assay.Finally, succinic acid production by C.acetoacidophilumΔldh showed that the metabolic flux to the lactic acid synthesis pathway of C.acetoacidophilumΔldh was zero, and the flux to the succinic acid synthesis pathway increased by 159%. C.acetoacidophilumΔldh could accumulate 54.4 g/L succinic acid with glucose and MgCO3 as the substrate, which yielded 43.6% and 35.8% more succinic acid than C.acetoacidophilum ATCC 13870 andⅡ-169, respectively.