Modification Of Phosphoenolpyruvate: Carbohydrate Phosphotransferase System Of Escherichia Coli By Genetic Engineering And Characterization Of Mutants

Phosphoenolpyruvate: sugar phosphotransferase system (PTS) plays an important role in phosphorylating and transporting glucose in Escherichia coli. A series of ptsG mutants and DH5α△ptsHIcrr were successfully constructed by using a one-step markerless deletion method that is a modified Red homologous recombination combined with specific cutting of I-SceⅠ. A liner targeting DNA fragment consists of two homing endonuclease sites at the both side of homology flanking which corresponed to the aim gene and finally correct contracted by enzyme cutting and connecting sequentially, Transform target plasmid and assistant plasmid into the host cell together, and express homing endonuclease and Red recombinase under induction by L-arabinose, as a result the in vivo recombination and gene substitution are carried out and DH5α△ptsG, JM109△ptsG, HB101△ptsG, Bl21(DE3)△ptsG and DH5α△ptsHIcrr were successfully constracted. In LB medium supplemented with glucose, the maximal cell densities of DH5α△ptsG, JM109△ptsG, HB101△ptsG, BL21(DE3)△ptsG were approximately 2.99, 3.38, 1.58 and1.70 times of their parents respectively and acetic acid of DH5α△ptsG, JM109△ptsG were obviously diminished. E.coli genome array shows that ptsG mutants have a wide influence on cell metabolism, EMP cycle and TCA cycle gets an balance, especially the cycle of glycolysis is weakened and tricarboxylic acid cycle is strengthened. In LB medium, DH5α△ptsHIcrr has a significant different characteristics of growth with DH5αand DH5α△ptsG, and the maximum biomass of DH5α△ptsHIcrr is about two times of that of DH5αor DH5α△ptsG. However, there was no difference between the growth of DH5αand that of DH5α△ptsG. In LB medium supplemented with 1% glucose, DH5α△ptsHIcrr and DH5α△ptsG both grew better than DH5α, and the maximal cell densities were approximately 2 times and 2.8 times of that of DH5αrespectively. In the end of culture, the concentrations of acetic acid of DH5α△ ptsHIcrr, DH5α△ptsG were12.2%, 47% of that of DH5α. In a modified M9 medium, the specific growth rate (h^-1) and the specific glucose consumption rate (g·g^-1·h^-1) of DH5α△ptsHIcrr were much slower than that of DH5αand a little slower than that of DH5α△ptsG. Altogether, the ptsHIcrr operon-deleted mutant changed the specific glucose consumption rate and showed many different characteristics of growth and metabolism from that of the△ptsG-deleted mutant. ptsG mutant of E.coli are available for high-density culture and the data of specific glucose consumption rate of DH5α△ptsHIcrr provides an important clew for further molecular modification especially can be used in production of shikimate pathway intermediates and amino acids.