Breeding Of L-Glutamine Producer And Study On Metabolic Control

L-glutamine (L-Gln) is an amino acid which has many special features. It is classified as conditionally essential amino acid. At present, mainly on the current clinical treatment, it is applied to cure depression, alcoholism and diseases such as stomach and duodenal ulcer. Additionally, it also can enhance nerve function, improve memory after brain hemorrhage barriers, promote poor intellectual development of children and prevent epileptic seizures and also many other functions. It is an extremely promising new drug. In this paper, three aspects such as the breeding of L-glutamine producing strain, the optimization of fermentation conditions and metabolic flux analysis, as well as the regulation of glutamine synthetase were studied.Firstly, the breeding of L-glutamine producing strain was studied systematically. The original strain Corynebacterium acetoacidophilum YL012 (MSOR) was mutated by the methods of UV and NTG treatments, and then it was screened by three resistant drugs: sulfaguanidine(SG), high concentration (NH4)2SO4 and sodium fluoride(NaF), meanwhile succinate as sole carbon source was also used. Finally one L-glutamine-producing strain LCHA082 (MSOR, SGR, (NH4)2SO4R, SAG, NaFR) was obtained. Without optimization there was an L-glutamine accumulation of 35.8 g/L, 9.4 g/L higher than the original strain.Secondly, medium component and fermentation conditions of mutant strain LCHA082 were optimized. The composition of fermentation medium, as well as the best conditions were as follows(g/L): glucose 130.2, (NH4)2SO4 58.7, corn steep liquor 2.78, KH2PO4 1.25, MgSO4·7H2O 0.6, MnSO4·4H2O 0.003, FeSO4·7H2O 0.005, ZnSO4·7H2O 0.006, CaCO3 30; 15mL/250mL, original pH 7.5, seed volume 8%, at 30℃with shaking at 100 r/min for 72 h. L-Gln formation was stable at 40.7 g/L, the maximum output was 42.4 g/L, which was 5.9 g/L improved after optimization.Thirdly, the center metabolic network of Corynebacterium acetoacidophilum YL012 and its mutant LCHA0082 were established and modified. The flow to node of glutamate increased from 29.198 mmol/L·h to 44.854 mmol/L·h, about 1.5 times of the original; The flow to L-glutamine synthesis changed from 18.138 mmol/L·h to 31.065 mmol/L·h, The analysis may play an important role in helping us to rationally re-design metabolism for further improvement of fermentation process.Finally, enzyme activity regulation of glutamine synthetase (GS) was carried out by way of nitrogen starvation-nitrogen gradient fed method. The conditions of nitrogen starvation were as follows: the beginning nitrogen was urea of 2.0 g/L, (NH4)2SO4 (20%) was fed nitrogen. And the best nitrogen starvation time was 15 h; fed method: add gradient four times, four fed concentrations were: 5 g/L, 10 g/L, 25 g/L and 15 g/L. Production could be 36.2 g/L after 48 h fermentation, L-Gln's mass ratio of the total could reach 75.6%. Metabolic flux analysis of nitrogen starvation was carried out to prove that the nitrogen starvation played a specific role on nitrogen regulation of glutamine synthetase activity.