Breeding Of L-Lysine Producer And Regulation Of Its Fermentation

According to the theory of metabolic control fermentation, the paper focused on the breeding of L-lysine hyper-producer LXQ-89, the metabolic fluxs of the original strain LXQ-8 and its mutant LXQ-81 and LXQ-89, the suitable fermentation condition of shaking flask and so on. The main research contents and results were as follows:The L-lysine producer was derived from the original strain Brebvibacterium flavum LXQ-8 by chemical and physical mutation methods, the plate screening with high concentrations of glucose, sulfa guanidine (SG), L-Lysine ethyl ester dihydrochloride (Lys-OEt) resistance, and valine (Val) defects.A strain LXQ-89 (Met-Thr-SucgAECrSGrVal- Lys-OEtr) was obtained and 77 g/L L-lysine was accumulated.The metabolic networks of the Brebvibacterium flavum LXQ-8, and the two derivatives carry additional mutations LXQ-81 and LXQ-89 were established and modified. The concentrations of extra-cellular metabolites were determined under sub-steady-state (42~46 h) of the batch culture. The metabolic flux distribution maps of the three strains were obtained, compared and analyzed. These results indicate that the introduction of analog supersensitive marker or deficient mutant marker skew the metabolic flux towards the formation of L-lysine. Metabolic flux analysis provided a new idea for design breeding rationally.The optimization of medium contents and fermentation conditions for LXQ-89 was conducted,acetic acid and ethanol were added into the fermentation medium,Tween-80 and dimethyl sulfoxide were added in the fermentation process. The optimum seed medium contained glucose 25 g/L, (NH4)2SO4 5 g/L, corn steep liquor 35 g/L, KH2PO4 1.0 g/L, MgSO4·7H2O 0.5 g/L, CaCO3 15 g/L. The optimum seed conditions were pH 7.0, and the optimum medium volume was 25 mL/250 mL. The optimum fermentation medium contained glucose 170 g/L, (NH4)2SO4 55 g/L, corn steep liquor 18.6 g/L, KH2PO4 1.2 g/L, MgSO4·7H2O 0.6 g/L, Fe2+ 2 mg/L,Mn2+ 2 mg/L, Met 0.4 g/L,Thr 0.4 g/L,Val 0.2 g/L, acetic acid 6.3 mL/L, biotin 100μg/L, VB1 200μg/L, CaCO3 45g/L, adding Tween-80 3.2 g/L at 20 h. The optimum fermentation conditions were inoculated time 11 h, original pH 7.0, seed volume 10%, 500 mL contains 25 mL broth, culturing at 30℃on reciprocating shaker, shaking speed 100 r/min, fermentation time 72 h. After optimization, the production of L-lysine was 95 g/L.The fed-batch fermentation was studied, combining with the fermentation process of stain LXQ-89. It was determined that the total glucose was 170 g/L, the initial glucose was 80 g/L, 45 g(for 1 L broth) glucose was added at 28 h, the remaining glucose was added at 44 h, and L-lysine reached 100 g/L at 72 h.