| Bacillus subtilis chungkookjang is a poly-γ-glutamic acidγ-PGA) producer screened from traditional Korean food, which can produce high molecular weightγ-PGA and has no any plasmid. In the present study, 3γ-PGA biosynthesis genes were cloned and employed to construct expression system in Eschericha coli. High cell density culture, DNA-microarray, Metabolic flux analysis were performed to optimize theγ-PGA production yield of the genetic engineering E. coli strains.The highestγ-PGA yield of Bacillus subtilis chungkookjang was achieved in the conditions that describe as following: 32g/L glucose, 48g/L glycerol, 14g/L NH4C1; 20g/L L-glutamic acid; 1% NaC1, initial pH 7.2, temperature 37℃, volume level 20 mL (250 mL flask), inoculum leve 15%. The yield was 8.2g/L in flask culture.The trc inducible expression vector pMpgs123 and the two constitutive expression vectors pCOpgs and pGntpgs were constructed with the threeγ-PGA biosynthesis genes (pgsl, 2, 3) from Bacillus subtilis chungkookjang. These three expression plasmid were transformed into E. coli host cells XL1-blue,BL21(DE3),W3110,FMJ123, BL21(DE3)-pCOpgs system achieved the highest production yield of 0.51g/L in flask culture. 2, 850 open reading frames (ORFs) of E. coli including all functional known genes were analyzed by DNA-microarray. Among the 2, 850 spotted genes, 196 genes were found to be up-regulated by above two-fold while 217 genes were down-regulated. The comparison transcript level of 18 genes relative to the glutamic acid metabolic pathway showed that the ceils were under the nitrogen starvation state during the production ofγ-PGA. 3.76g/Lγ-PGA was achieved when 40g/L (NH4) 2SO4 was added into the 600g/L glucose feeding solution of the pH-stat fed-batch culture of BL21(DE3)- pCOpgs. The flux distribution of a-KG to sucCoA was larger than the distribution to L-glutamic acid, which might be the primary limiting factor for optimization of poly-γ-glutamic acid production in the genetic engineering E. coli.
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