Study On The Fermentation Of L-Valine With Corynebacterium Glutamicum

In this paper, the methodologies of fermentation principles and metabolic engineering wereapplied into the strain improvement of Corynebacterium glutamicum AATV341for the overprod-uction of L-valine. The effect of acetate as a glucose co-substrate on growth, valine synthesis fromcarbon substrates by Corynebacterium glutamicum AFTS134 was investigated. Thetemperature-triggered process also used to improve valine-producing process.Paper chromatography-spectrophotometry was applied into determine the L-Val in btoth wasstrdied, the conditions in the course of determining the L-Val was established, the reclaim ofaverage percentage of this method was 100.7%, so this method is accerate to determine theconcentration of L-Val in btothBased on the strategies of increasing the concentration of pyruvate, precursor of valine, threemutants were obtained by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis anddirectional screening derived from the strain Corynebacterium glutamicum AATV341. We alsoensure the optimal concentration of biotin and VB₁. The mutant AFTS134 derived from AATV341,AT22 ( Leu-,2-TA^r ) and AFT31(Leu-,2-TA^r,3-FPYR^SS) was obtained with four genentic markers:Leu^-,L-FPYR^ss,2-TA^r (1 g/L),Suc^g ( grow quickly on medium using succinate as only carbonsource ). The original strain, Corynebacterium glutamicum AS1.495 ( Leu^-), can produce 10.5g/Lof L-valine. By strain AFTS134, 22.4 g/L of L-valine was produced stably, which increased 1.18times more than AATV341.The effect of the mixed substrate(glucose-acetate) medium compositions and cultureconditions on L-valine production was investigated. According to above experimental results, thebetter medium compositions were determined as: glucose 90 g/L,(NH₄)₂SO₄ 40 g/L,KH₂PO₄ 1g/L,MgSO₄·7H₂O 0.5 g/L,Fe_SO₄·7H₂O 2 mg/L,MnSO4·H₂O 2 mg/L,biotin 20μg/L,VB₁ 200μg/L,soybean hydrolysate 20 g/L,CaCO₃ 50 g/L, pH 7.2,and the better fermentationwas performed at 30℃ on a rotary shaker (160 r/min )with 20 mL culture broth inoculated by 10percent. AFTS134, adding 0.02g acetate after 24 h and measure the concentration of valine after 64h. produced 28.4 g/L of L-valine under above conditions, which increased 40% more than before.With the existing knowledge in biochemistry, genetics and physiology of Corynebacteria, thetemperature-triggered process was investigated for valine. By a tight control of the cultivationtemperature, it was possible to get interesting performance. A 64 h fermentation period enabled theproduction of 23.5 g/L of L-valine in the production phase induced after a temperature shift from30℃ to 37℃ at 32 h. It increased 11% more than before. In another experiment we added 0.02 gacetate to medium when the shifting had happened. We found there was no change in theconcentration of valine but the concentration of glutamate increased by 5 times...