Construction And Fermentation Optimization Of E.coli For High-producing Succinate

Succinate,as an important platform chemical with four carbon atoms,has broad applications in chemical,pharmaceutical,agricultural and food fields.Based on sustainable development,microbial fermentation of succinate has been attracted more and more attention.The wide-type Escherichia coli FMME-N can naturally produce succinate.However,its titer,yield and productivity was too relatively low to meet the criterion of industrial production.In this study,we metabolically engineered E.coli FMME-N strain by combinatorial mutagenesis,metabolic engineering（including gene knockout and cofactor regulation）,and fermentation optimization for improving the production of succinate.The main contents are as follows:1.Screen of mutants with high osmotic-tolerant.Firstly,the productivity of strain E.coli FMME-N was decreased to 0.54 g?L^-1?h^-1 with the increasing of the osmotic pressure,which indicated that osmotic pressure might be one of the limiting factors for succinate.Secondly,strain FMME-N was mutagenized and screened by combining ARTP and ⁶⁰Co-γrays with 0.9 M Na Cl as a screening inhibitor.Then,the mutant strain E.coli FMME-N-2 that could tolerance to 2192 m Osmol?L^-1 osmotility was obtained.Finally,the titer,yield and productivity of succinate were 51.8 g?L^-1,0.70 g?g^-1glucose and 0.81 g?L^-1?h^-1 in 3.6 L fermentor,which increased 45.5%,12.9%and 51.7%compared with that of the strain E.coli FMME-N,respectively.2.Block of competitive metabolic pathways.In order to improve the succinate titer,several genes were knocked out,including lactate dehydrogenase（ldh A）,pyruvate formate lyase（pfl B-foc A）,phosphotransacetylase（pta）and acetate kinase（ack A）.Among them,the production of lactate and formate were decrased in strain FMME-N-10（FMME-N-2Δpfl B-foc AΔldh AΔpta）,but the titer of acetate was increased.Then,strain FMME-N-13（FMME-N-10Δtdc DE）was constructed by further knocking out the propionate kinase（tdc D）andα-ketobutyrate formate lyase（tdc E）.The results showed that the succinate titer,yield and productivity in E.coli FMME-N-13 were 89.6 g?L^-1,0.92 g?g^-1glucose and 1.40 g?L^-1?h^-1,respectively,with an increased of 72.9%,31.4%and 72.8%compared to strain FMME-N-2.Meantime,the accumulation of lactate,formate and acetate were reduced by 86%,52.6%and 28.9%compared with the strain FMME-N-2.3.Construction of cofactor regeneration system.Based on above results,the yield of succinate was 0.92 g?g^-1_glucose compared with the theoretical succinate yield of 1.12g?g^-1_glucose,which indicated that inadequate intracellular cofactor supply was a limiting factor for succinate production.Based on this,phosphoenolpyruvate carboxykinase（As PCK）from Actinobacillus succinogenes and formate dehydrogenase（Cb FDH）from Candida Boidinii were overexpressed for improving cofactor level and constructed the strain FMME-N-14 and FMME-N-15,resulting succinate titer increased by 11.1%and8.3%,respectively.Then,the expression level was optimized using different strengthes of RBS.The results showed that the strain FMME-N-26（FMME-N-13-L-As PCK-L-Cb FDH）was constructed when these two enzymes expressed in low level,contributing to the intracellular NADH and ATP were increased while the NADH/NAD⁺ratio remained at a relatively stable level,which resulting in the succinate titer,yield and productivity were 101.4 g?L^-1,1.05 g?g^-1glucose,and 1.58 g?L^-1?h^-1 in 3.6 L fermentor,with an increase of 13.2%,14.1%,and 12.9%than the strain FMME-N-13,respectively.4.Optimization of fermentation conditions.In order to further improve the yield of succinate,the two-stage fermentation process was optimized in strain FMME-N-26from three conditions including the transition time of two-stage fermentation,p H neutralizer and glucose concentration in anaerobic fermentation broth.The results showed that when grew to OD₆₀₀=30 then turned to anaerobic fermentation,magnesium carbonate was used to maintain the p H and mataining<5 g?L^-1 glucose concentration of the fermentation broth,resulting in the titer,yield and productivity of succinate were increased to 139.2 g?L^-1,1.11 g?g^-1glucose and 2.18 g?L^-1?h^-1.Finally,the titer,yield and productivity of succinate were 136.8 g?L^-1,1.11 g?g^-1glucose and 2.14 g?L^-1?h^-1 under the optimum conditions above in 30 L bioreactor.Only 2.2 g?L^-1 lactate and 5.1 g?L^-1acetate were detected in fermentation broth.