Breeding Of L-glutamic Acid Producer And Study Of Its Metabolic Control

The original strain Corynebacterium glutamicum TWQ080 were treated gradually by mutagen, and a L-glutamic acid producer GW46-4 was obtained based on metabolic engineering principle. Then, fermentation conditions in flask and fermentor were studied, and the metabolic flux distribution of original strain and mutant were also studied. The research and its main results are as follows:(1) The original strain Corynebacterium glutamicum TWQ080 was treated gradually by UV and DES and then was screened by high concentrations of glucose and glutamate.As a result, a L-glutamic acid producing strain GW46 was obtained. The thermotolerant property of Corynebacterium glutamicum was improved gradually via a passage transfer approach. After several times of domestication, a thermotolerant strain GW46-4 was obtained. An accumulation of 92.5g/L glutamic acid was obtained in the original culture condition, which was 31.2% higher than the original strain.(2) The seed medium and culture conditions were studied. It was found that the optimal medium components were glucose 25g/L,corn steep liquor 30g/L, K2HPO4·3H2O 1.5g/L, urea 6g/L, MgSO4·7H2O 0.4g/L and the optimal culturing conditions were initial pH7.0, medium volume 30mL/250mL. The fermentation medium and conditions in shaker flask were studied. The optimal medium components was glucose 158.3g/L, corn steep liquor 3.2g/L, K2HPO4·3H2O 1.5g/L, MgSO4·7H2O 0.8g/L, MnSO4·4H2O 20mg/L, FeSO4·7H2O 20mg/L, VB1200μg/L. The optimal fermentation conditions were medium volume 20mL/500mL, inoculated volume 8%, seed age 7h. L-glutamic acid concentration reached 102.6g/L in flask, which was 10.9% higher than before optimization.(3) The fed-batch fermentation was studied. It was found that the optimal initial glucose was 100g/L and the residual glucose concentration was 10-20g/L. The fermentation in fermentor was also studied and found the optimal DO control strategy was controlled at 30-40%. The optimal temperature control mode was 0-8h at 32℃,8-16h at 34℃,16-30h at 36℃. In 7L fermentor, L-glutamic acid concentration was 114.2g/L, which was 23.5% higher than before optimization. In addition, the conversion rate was 59.4%.(4) The metabolic network of Corynebacterium glutamicum was established and the metabolic flux distribution of the original strain and mutant were studied. It was found that the CO2 fixed equation was strengthened and the glyoxy cycle was decreased. The flux to glutamic acid from the node of isocitrate was increased. Compared with the original strain, the flow to synthesis of other amino acids by using glatamic acid as precursor was decreased.