Construction of a Novel Engineering Strain E.coli G830,which is Adoptable to High-cell-density Fermentation

A novel engineered strain G830 adoptable to high-cell-density fermentation by integrating bacterial hemoglobin vhb (Vitreoscilla hemoglobin gene) into thr operon in the chromosome of PA1 blocking Pta-Ack metabolic pathway through the homologous recombination between the homologous fragments of integrated plasmid and that of chromosome. The engineered strain G830 was characterized by phenotype observation, PCR, thr mutant, acetate acid detection, Western blotting and VHb activity assays. In high dentity fermentation, the cellular respiration, energy metabolism, highest bacterial density and dry bacteria weight of the G830 strain were markedly better than control strains PA1 and BL21. The expression of recombinant prolyl endopeptidase (PEP) in G830 and PA1 under the above condition was high and stable. Their growth situation and fermentation parameters were similar with their parental strains without plasmids, and resided plasmids maintained stably in those strains. It revealed that the integral VHb and acetate metabolism pathway (Pta-Ack) block improved the growth of host strain under low-dissolved-oxygen conditions, enhanced the recombinant proteins production and reduced the accumulation of acetate harmful to bacterial growth. In conclusion, the novel engineering strain G830 was adoptable to high cell density fermentation.