Effects Of PtsG Knockout On Carbon And Nitrogen Metabolism Of Recombinant Escherichia

It was well known that the unbalanced metabolisms of carbon and nitrogen usually resulted in high accumulation of by-products, low yield of the target products, and even the failure of fermentation process. In this work gene knock-out and metabolic modification were introduced to balance the metabolisms of carbon and nitrogen and the recombinant E. coli BL21 3.7 kptsG was constructed. The effects of ptsG deletion on glucose metabolism and ammonium metabolism were investigated at transcriptional level and enzymatic level in this work. The results show that ptsG deletion affected not only glucose transport directly but also glucose metabolism indirectly. Due to the formation of global regulator cAMP-crp and the multilevel mediations by cAMP-crp and Cra, ptsG deletion enhanced the activities of pyruvate kinase and phosphoenolpyruvate carboxylase remarkably, and the transcription level of ppc coding for phosphoenolpyruvate carboxylase was 1.23-fold higher than that of its parent strain. These changes regulated recombinant E.coli to use glucose in a balanced way accompanying a decrease of acetate excretion and the transcriptional level of ackA was 0.30-fold lower than that of its parent strain and the acetate accumulation decreased by 76.3%. As to ammonium metabolism, the effects of ptsG deletion on nitrogen metabolism were similar to lowering C/N ratio, in order to match a lower carbon flux, the activity of GS was downshifted while that of low-affinity enzyme GDH was upshifted, the expression level of gdhA was increased too (1.88-fold higher than that of its parent strain) and GDH acted as the main pathway for assimilating nitrogen in ptsG’strain so as to match the glucose metabolism.Then the growth characteristics of HLC recombinant E.coli and itsptsG mutant cultivated in the modified minimal medium containing different carbon source (glucose, xylose, and its mixed sugar) were investigated. The results show that E. coli BL21 3.7 AptsG showed stronger ability to produce target protein in the medium with glucose as the solo carbon source. The yield of HLC production of ptsG mutant reached 0.27g/L, increasing by 17.3%, compared with that of its parent strain (0.23g/L); ptsG deletion hadn’t dramatically affect the metabolism and the yield of HLC production in the medium with xylose as the solo carbon source. The yield of HLC production of ptsG mutant reached 0.18g/L, nearly the same as its parent strain (0.19g/L); And E. coli BL213.7 △ptsG can simultaneously utilize glucose and xylose, enlarging the scope of available carbon source and the HLC productivity in the medium with mixed sugar as carbon source. And the yield of HLC production of ptsG mutant reached 0.20g/L, increasing by 11.1%, compared with that of its parent strain (0.18g/L).