Study On Succinate Production By Metabolic Engineered Corynebacterium Acetoacidophilium

As one of the most important C4 block platform chemicals, succinate has always been used in food, pharmaceutical and chemical industry. With the development of modern chemical industry, bio-based succinate bioprocess has the potential to replace chemical-based process in large scale. An ldh A gene deficient strain of Corynebacterium acetoacidophilum was constructed and the capacity of producing succinate was studied in the previous work in our lab. However, some limits such as the low titer of succinate, high ratio of acetate and low rate of glucose consumption rate still existed. Here, we focused on the metabolic engineering of C. acetoacidophilum as well as the two-stage process of succinate production.Fristly, the effects of disrupting acetate formation pathways in C. acetoacidophilum Δldh A on succinate production were studied. Mutants of C. acetoacidophilum Δldh AΔpta-ack A, C. acetoacidophilum Δldh AΔpta-ack AΔctf A and C. acetoacidophilum Δldh AΔace E were constructed with knocking out genes pta, ack A, ctf A and ace E using the method of homologous recombination, respectively. The results of succinate production under oxugen deprivation showed that the acetate titer, glucose consumption rate and yield of succinate were not changed significantly after the deficience of gene pta and ack A. The simultaneous deficience of gene pta, ack A and ctf A resulted in the 81.4% and 77.2% decrease of acetate titer and yield respectively, 28.3% decrease of glucose consumption rate and 25.3% increase of yield of succinate. However, almost no acetate was excreted accompanied by 35.6% decrease of glucose consumption rate and 34.7% increase of yield of succinate when only gene ace E was deficient. Thus, the preliminary acetate formation pathways in C. acetoacidophilum were achieved. The disruption of acetate formation pathways is helpful to increase the yield of succinate, but could decrease the glucose consumption rate.Secondly, in order to improve the glucose consumption rate and succinate production, the overexpression of gene gap A encoding phosphoglyceraldehyde dehydrogenase and gene pyc encoding pyruvate carboxylase in C. acetoacidophilum Δldh A were studied. The results showed that the glucose consumption rate of strain C. acetoacidophilum Δldh A/p XMJ19-gap A increased 16.2% compared with C. acetoacidophilum Δldh A, which was also far away from that of wild-type strain C. acetoacidophilum ATCC13870. Strain C. acetoacidophilum Δldh A/p XMJ19-pyc could excrete 60.5% more succinate in titer and yield compared with C. acetoacidophilum Δldh A and 183% more than C. acetoacidophilum ATCC 13870. The overexpression of pyc in strain C. acetoacidophilum Δldh AΔace E could increase the yield and titer of succinate by 43.8% compared with C. acetoacidophilum Δldh A and 7.4% compared with C. acetoacidophilum Δldh AΔace E. Among all the mutants, strain C. acetoacidophilum Δldh A/p XMJ19-pyc got the best potential of producing succinate.Finally, the bioprocesses of succinate production by strain C. acetoacidophilum Δldh A/p XMJ19-pyc were studied. Growth-arrested bioprocess was used in fed-batch conversions and recycle of concentrated cells. After converting for 80 h in flask, 90 g·L-1 succinate were accumulated with a yield of 0.81 g·g-1(succinate/glucose). 121 g·L-1 succinate was excreted in 3-L fermenter in 75 h with a yield of 0.85 g·g-1. The cells in 3-L fermenter were recycled in batch conversion to produce succinate. But results showed the glucose consumption rate and succinate titer decreased due to the reduction of cell concentration and vitality. An improvement method with application of two-stage bioprocess was carried out. In 500 m L flask, C. acetoacidophilum Δldh A/p XMJ19-pyc could accumulate 94.2 g·L-1 succinate after converting for 102 h. When enlarged in 3-L fermentor, 107 g·L-1 succinate was excreted in 96 h with a yield of 0.88 g·g-1(succinate/glucose) which incates the feasibility of operation.