Breeding Of L-arginine Producer And Initial Study Of Its Metabolism

In this dissertation, breeding of the L-arginine producing strain, optimization of fermentation conditions, as well as metabolic flux analysis were systematically studied based on the fermentation principles of metabolism control. The main research contents and results are as follows:⑴The L-arginine producer YHR-109 (SGR, Sucg, L-HAR) was obtained from the original strain Corynebacterium glutamicum ZGH-064 by stepwise mutagenic treatments with ultraviolet rays (UV), diethyl sulfate (DES) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). In the strain screening process, sulfaguanidine (SG), L-homoarginine and succinate (Suc) were used to produce selecting medium respectively. The final mutant YHR-109 could accumulate 15.5g/L L-Arginine before culture optimization.⑵The effects of seed medium compositions on the L-Arginine production in flasks were investigated using orthogonal analysis. The optimum seed medium compositions were determined as follows: glucose 25g/L, (NH4)2SO4 15g/L, corn steep liquor 25g/L, KH2PO4 1g/L, MgSO4·7H2O 0.5g/L, CaCO3 20g/L, pH7.0; and the optimum culture volume was 25mL/250mL.⑶With the help of the software Design Expert, a response surface model was built using the Central Composite Design to optimize the fermentation medium. Prior to the RSM experiment, 4 single-factor experiments: glucose, ammonium sulfate, corn steep liquor and biotin were analyzed. The results showed that, the optimum conditions for YHR-09 to produce L-arginine were as follows: glucose 110.0g/L, corn steep liquor 23.7g/L, (NH4)2SO4 48.0g/L, KH2PO4 1.5g/L, MgSO4·7H2O 0.5g/L, biotin 80μg/L, CaCO3 30g/L, pH 7.0. In the optimum fermentation medium, the mutant YHR-109 which was cultivated 96h at 30℃on a reciprocating shaker (100r/min) with 15mL medium in the flask (250mL) could produce 21.0g/L of L-Arginine on average, which increased about 35.4% more than before.The result of the comparing experiment showed that the improvment of the nutritional conditions of the medium made one of the main contribution to the increase of the L-arginine production.⑷The simplified model of the L-Arginine biosynthetic network was constructed based on the theory of metabolic flux analysis. Metabolic flux distribution of L-arginine biosynthesis in the strain YHR-109 were calculated using the software Matlab and the flow of PYR-node and Glu-node which have main effects on the synthesis of L-arginine were analysed. The results showed that the flux phosphoenolpyruvate to ketosuccinic acid is very small (only 11.65), which may be the main cause of the small flux (only 9.15) of L-arginine synthesis. Thus, in the next breeding practice, strengthening the CO2 fixation could be considered. In addition, as L-glutamic acid participates in the biosynthesis of many amino acids as the amino donor, the synthesis of other amino acids results in a large amount of invalid flow cycle between theα-ketoglutarate and L-glutamate. So it is meaningful to further improve the level of resistance of the stain to L-arginine analogues in order to reduce the miscellaneous acids.