Breeding Of L-glutamic Acid Producer And Study Of Conditions And Metabolic Flux In Fermentation

According to the theory of metabolic control fermentation, the producing of L-glutamic acid by fermentation using Corynebacterium glutamicum WH063 as original strain has been studied in this dissertation. The paper focuses on the breeding of L-glutamic acid hyper-producer WH063. Then the fermentation conditions were optimized. The metabolic fluxs of the original strain WH063 and its mutant TWQ080 were studied. The main research contents and results are as follows:The original strain Corynebacterium glutamicum WH063 by stepwise mutagenic were treated with diethyl sulfate (DES), ultraviolet rays (UV) and N-methyl-N'-nitro-N- nitrosoguanidine (NTG). The plate screening with amino acid analogues: sulfaguanidine (SG), propanedioic acid (PA), Fluoropyruvate (FP) sensitive and used succinic (SA) as sole carbon sourse. A strain TWQ080 (SGr, Pror, Sucg, FPs) which could accumulate L-glutamic acid 92.5g/L in original medium.The medium contents and fermentation conditions for TWQ080 was investigated by using orthogonal and response surface analysis. Corn steep liquor and K2HPO4·3H2O were more influence to the seed quality. The optimum seed medium compositions were determined as follows: glucose 25g/L, corn steep liquor 35g/L, K2HPO4·3H2O 1.5g/L, MgSO4·7H2O 0.4g/L, urea 5g/L. The optimum fermentation medium contains glucose 180g/L, corn steep liquor 3.07g/L, K2HPO4·3H2O 2.95g/L, MgSO4 1.10g/L.The optimum fermentation conditions were 500mL contains 15mL broth, seed volume 6%, inoculated time 8.5h, culturing at 30℃on reciprocating shaker, shaking speed 100r/min, fermentation time 38h. After optimization, the production of L-glutamic acid was up to103.1g/L. conversion rate57.6%. TWQ080 strains flask fed-batch fermentation of the preliminary was studied. The optimum initial glucose concentration was 100g/L. The lowest glucose concentration maintained was 20-30g/L. The production of L-glutamic acid was up to 106.5g/L, which increased about 34% more than before. And conversion rate was up to 59.8%.The simplified model of the L-glutamic acid biosynthetic network was constructed based on the theory of metabolic flux analysis. The metabolic networks of the Corynebacterium glutamicum WH063 and its mutant TWQ080 were established and modified. The concentrations of extra-cellular metabolites were determined under sub-steady-state (24-26h) of the batch culture. The metabolic flux distribution maps of the original strain and its mutant were obtained, analyzed and compared. The flux partitioning at pyruvate node changed greatly in WH063 and its mutant TWQ080. The flux to the pathways of L-glutamic acid from the pyruvate node, which was 58.963mmol·L-1·h-1 in the original strain, reached 63.888 mmol·L-1·h-1 in the strain TWQ080 and the flux to the"other acids"and"outgrowth"from the pyruvate node, which was 70.260mmol·L-1·h-1 in the original strain, changed to 66.696 mmol·L-1·h-1 in the strain TWQ080. L-glutamic acid as precursor biosynthetic"other acids"the flux which was 0.333mmol·L-1·h-1in the original, reduced to 0.057mmol·L-1·h-1 in TWQ080.