Improvement And Recombination Of E.Coli With Metabolic Engineering

The vgb gene was integrated and ptsG gene was knock-out with Red homologous-recombination. The chloramphenicol-resistant gene and vgb gene with two flanks homologous with ptsG gene's were PCR-generated. After electroporated into Escherichia coli DH5α and JM109, the chloramphenicol-resistant gene and vgb gene took the place of ptsG gene with the help of Red recombinase. In LB media supplemented with glucose, the mutants of Escherichia coli showed greater biomass and recombinant protein productivity than that of the wild-type strains, together with exploiting more glucose. The amount of acid excretion has been reduced in the mutants. The results demonstrate that the mutant strains are available for high cell-density culture. N-carbamoylase was expressed with pBV220 in DH5αPV greater than in DH5α.The cell wall protein of Bacillus brevis DY50 was purified through aion-exchange chromatography and hydrophobic chromatography. Antibody was prepared by immuning rabbit, whose value was 1:50000. A genomic library of Bacillus brevis DY50 was constructed with phage arm and was screened with dot-ELISA which provides basis work for attaining the strong promoter of cell wall protein to construct the express system in the mutant Escherichia coli. N-carbamoylase was expressed with the strong promoter of α-amylase in DH5α.