Deletion Of AdhE,ldhA,ackA In Chromosome Of Ecoli By Red Recombination

Plasmid pKD46 can express three proteins: Gam, Bet and Exo. Gam inhibitsthe host RecBCD exonuclease V, so that Bet and Exo can gain access to DNA endsto promote recombination. BW25113 with pKD46 has the function of recombinationwhen induced by L—arabinose. PCR products were obtained by using primers with39bp extension which were homologous to adhE,ldhA,ackA and by using templateplasmid named pKD3(or pKD4) carrying chloramphenicol(or kanamycin)resistancegene flanked by FRT sites. The PCR products were introduced into BW25113 byelectroporation. The strains expressing chloramphenicol resistance gene wereselected bychloramphenicol agar. The chloramphenicol(or kanamycin)resistancegene was then eliminated by using a helper plasmid,pCP20, encoding the Flprecombinase. Using this system, adhE,ldhA gene in chromosome of BW25113D wereall deleted, and ackA gene in chromosome of BW25113 was also deleted. Therecombinant plasmid pSE-ldh expressed the L-(+)-lactate dehydrogenase ofStreptococcus bovis was transformed into E. coli BW25113D. The SDS-PAGE show thegene can express in the BW25113D. The activity of this gene was 150U/mL. TheBW25113D/pSE380-ldh was Shake-flask Ferment-cultivating for 35 hours, using theHLPC to detect the fermentation products, but found no L-lactate.