Study On Production Of L-arginine By Microorganism

According to the theory of metabolic control fermentation, the paper focuses on the breeding of L-arginine hyper-producer GUI089, the metabolic fluxs of the original strain and its mutant GUI089, the suitable fermentation condition of shaking flask and so on. The main research contents and results are as follows:The L-arginine producer was derived from the original strain Corynebacterium glutamicum GWY020 by chemical and physical mutation methods, the plate screening with amino acid analogues: arginine methyl ester HCl, sulfaguanidine, D-arginine, proline sensitive and used succinate as sole carbon sourse.A strain GUI089 (AEr, SGr, Sucg, D-Argr, Pros) which could accumulate 19.27g/L L-arginine was acquired in medium using glucose as carbon source and ammonia sulphate as nitrogen source.The metabolic networks of the Corynebacterium glutamicum GWY020 and the two derivatives carry additional mutations HUI821and GUI089 were established and modified. The concentrations of extra-cellular metabolites were determined under sub-steady-state (50~52h) 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 analog resistant marker skew the metabolic flux towards the formation of L-arginine. This study revealed the usefulness of the metabolic flux analysis as a tool for verification of existing production strains. The analysis may play an important role in helping us to rationally re-design metabolism for further improvement of fermentation process.The optimization of medium contents and fermentation conditions for GUI089 was conducted. The optimum seed medium contains glucose 25g/L, (NH4)2SO4 25g/L, corn steep liquor 25g/L, KH2PO4 1.0g/L, CaCO3 20g/L, MgSO4·7H2O 0.5g/L, pH 7.0; and the optimum medium volume was 25mL/250mL. The optimum fermentation medium contains glucose 99.11g/L, (NH4)2SO4 37.63g/L, corn steep liquor 20.90g/L, KH2PO41.5g/L, biotin 80μg/L, MgSO4·7H2O 0.6g/L, CaCO3 25g/L. The optimum fermentation conditions were 250mL contains 15mL broth, original pH7.2, inoculated time 18 h, culturing at 30℃on reciprocating shaker, shaking speed 100rpm,seed volume 8%,fermentation time 90h.After optimization,the production of L-arginine was up to 21.70g/L.Combining with the fermentation process of stains GUI089, the fermentation process was studied by adding some nutrients. It showed that the amount of L-arginine increased to different extent with proper way of adding glucose, glutamic acid and amino.Based on the metabolic network and fermentation of Corynebacterium glutamicum GUI089, the metabolic network of L-arginine was regulated with the addition of amino acid, organic acid and vitamin. The result showed most kinds of additives could influence the acceleration of L-arginine.Among the additives used in this study, L-glutamine, L-leucine, L-aspartic acid, L-valine, L-arginine, oxalic acid, malonicacid, amber acid, nicotinamide, pyridoxine, folacin and thiamine could improve the production of L-arginine. The yield of L-arginine was increased by 10% compared to the experiment without additives. Lower production of L-arginine was found following the addition of peptone, beef extract, and so on. By contrast, more L-arginine was produced after addition of SDS, chloramphenicol, yeast extract and soy bean protein hydrolazate. Tween 80, sodium phytate inhibited the growth of cells and induced decreases in the production of L-arginine.From the metabolic perspective, these additives not only improved the growth of the strain, but also avoided the overproduction of the additives. Therefore, the metabolic path of L-arginine production by GUI089 was strengthened.