Breeding Of L-arginine Producer And The Optimization Of Its Fermentation Conditions

In this paper, the fermentation principles of metabolism control were applied into original strain improvement of ZH-84 for the overproduction of L-Arginine, which was separated from Corynebacterium glutamicum UNC90. Then the fermentation conditions were optimized. The metabolic fluxs of the original strain and its mutant were studied. The main research contents and results are as follows:1. The L-Arginine producer was derived from the original strain Corynebacterium glutamicum ZH-84 by stepwise mutagenic treatments with diethyl sulfate (DES), ultraviolet rays (UV) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). In the plate screening, sulfaguanidine (SG), Arginine methyl ester HCl (AE), D-Arginine (D-Arg) and Cysteine (Cys) were used as amino acid analogues and succinate (Suc) as sole carbon source. The final mutant ZH5-82 could accumulate 16g/L L-Arginine that was about three times as the quantity produced by the original strain.2. The 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 30g/L, (NH₄)₂SO₄ 20g/L, corn steep liquor 20g/L, soybean protein hydrolyzate 10g/L, KH₂PO₄ 1.0g/L, MgSO₄·7H₂O 0.5g/L, CaCO₃ 10g/L, pH7.0; and the optimum inoculation volume was 6%.3. The effects of fermentation medium compositions were also studied with two-level orthogonal design and response surface analysis. The optimum fermentation medium compositions were determined as: glucose 90g/L, (NH₄)₂SO₄ 30g/L, corn steep liquor 25g/L, KH₂PO₄ 1.0g/L, MgSO₄·7H₂O 0.5g/L, biotin 80μg/L, CaCO₃ 30g/L, pH7.0. And in the optimum fermentation medium, the mutant ZH5-82 which was cultured 96h at 30℃on a reciprocating shaker (100r/min) with 15mL medium in the flask (250mL) could produce 18.3g/L of L-Arginine on average, which increased about 13% more than before.4. The effects of various substances on L-Arginine production had been investigated. Improvements were observed when 1g/L L-Glu, L-Leu, L-Val, L-Lys, L-Gly were added in medium. Of organic acids, succinate could enhance the L-Arginine production slightly. Nicotinic acid, folic acid, VB6 and low concentration thiamin could increase the quantity of L-Arginine in fermentation broth. Lower production of L-Arginine was found following the addition of alcohols and surfactants. Beef extract, peptone, yeast extract and soybean protein hydrolyzate didn't affect the production of L-Arginine apparently.5. The fed-batched cultivations with glucose, ammonia, urea and L-Glutamate were studied. It was found that the quantity of L-Arginine could be improved about 15% by continuous additions of glucose and ammonia into the fermentation broth. The continuous addition of urea didn't affect the production of L-Arginine apparently. The precusor L-Glutamate that was added during the mid and later periods could improve L-Arginine level.6. The simplified model of the L-Arginine biosynthetic network was constructed based on the theory of metabolic flux analysis. The maximal stoichiometric theoretical yield of L-Arginine...