Breeding Of L-Valine Producer And Regulation Of Its Fermentation Conditions

In order to improve the yield and quality of L-valine by microorganism in fermentation industry, two strategies including breeding high production activity strains and optimizing the fermentation conditions should be taken into account. The metabolic control fermentation breeding has become an important strain improvement strategy. In this paper, breeding of L-valine producer NJ-5 was conducted. Then, fermentation conditions were optimized and the metabolic flux distribution of the three L-valine producers was studied. The main results are as follows:(1) A strain NJ-2 was screened on high-glucose gradient plates by fusing the single inactivated protoplasts between Brevibacterium flavum NV128 and JV16 (Leu-Ile-Met-). L-valine production of the fusant was 42.3g/L for 72h in shaker flask. At the same time, three crucial conditions (penicillin, lysozyme and osmotic stabilizer) affecting formation and regeneration of protoplasts were studied, and the optimum treatment concentrations and time were determined.(2) The strain Brevibacterium flavum NJ-2 was treated by gradual high-glucose adaptive culture, synchronized enrichment culture, and then by stepwise mutagenic treatments with ultraviolet rays (UV) and diethylsulfate (DES). As a result, a L-valine producing mutant NJ-5 (Glchrα-ABr 2-TAr) was obtained, which could be resistant to high concentration glucose,α-aminobutyric acid(α-AB) and 2-thiazole-DL-alanaine(2-TA). The strain NJ-5 could accumulate 47.2g/L L-valine before culture optimization.(3) The seed medium ingredients for NJ-5 were optimized by orthogonal design test. The optimal seed medium ingredients were as following (g/L):glucose25, KH2PO4 1, (NH4)2SO4 3, MgSO4·7H2O 0.5, corn steep liquor 40, CaCO3 10.The optimal seed medium volume and Initial pH was 25mL/250mLand 7.0, respectively.(4) The medium ingredients and fermentation conditions for NJ-5 were studied. On the basis of single-factor experiments, a response surface model with the software Design Expert was built to optimize the fermentation medium. The results showed that the optimum medium for NJ-5 were as following (g/L):glucose 135, (NH4)2SO4 45, corn steep liquorl0.1, KH2PO4 1.2, MgSO4·7H2O 0.6, biotin 75μg/L, vitaminB1 90μg/L,CaCO3 30. The optimal fermentation conditions were initial pH 7.0～7.5, seed volume 5%. Under the optimal conditions, the strain NJ-5 which was cultivated for 72h could produce L-valine 52.3g/L on average. L-valine production of the strain was 53.8g/L in the 7 L fermenter.(5) The metabolism network model of the two original strains arid NJ-5 were established and modified, and the metabolic flux distribution of main nodes of three strains were analysed. The results showed that the flow to node of pyruvic acid was increased, and the flow to TCA cycle and complement reaction of TCA cycle was decreased. The metabolic flux was redistributed greatly for the biosynthesis of L-valine. The conclusion was that metabolic flux was detected for the biosynthesis of L-valine effectively by the combination use of several breeding methods. These results provided a novelty way for next re-design strain breeding.