Knockout Of The PtsG Gene In Escherichia Coli And Culture Characterization Of The Mutants

By means of Red homologous-recombination, the ptsG gene for glucose-specific transporter Enzyme II CB^Glc of the phosphotransferase system was knock-out so as to reduce the accumulation of acetic acid in the high cell-density culture of Escherichia coli on excess glucose. The chloramphenicol-resistant cassette was PCR-generated with short shared sequences on the both ends.After electroporated into Escherichia coli DH5a and JM109, the chloramphenicol-resistant cassette took place of ptsG gene with the help of Red recombinase to construct the mutants called DH5aP and JM109P. In LB media supplemented with glucose, the mutants of Escherichia coli deficient in ptsG showed greater biomass and less decline in pH, together with exploiting more glucose.The products of TNF were respectively 47.1% of total cellular protein in strain DH5aP and 40.9% in strain JM109P. These results demonstrate that the mutant strains will be available for high cell-density culture.