Abstract: Objective:In order to develop a gene replacement method that based on the Red recombination system of E. cloacae. Method:pSC-MSC-red and pSC-MSC-flp was constructed with the Red recombinase genes and Flp recombinase respectively. BudA encoded α-acetylactate decarboxylase was used as a target for gene replacement. The disruption cassettes with different lengths of homologous extensions were constructed. E. cloacae/pSC-MSC-red was transformed with these disruption cassettes for gene replacement. Results:Disruption cassettes with 39 or 100 bp homologous extensions fail to get replacement. E. cloacae ΔbudA-773 was obtained with disruption cassette hold 200 bp homologous extensions,and the efficiency of gene replacement was 6.1 CFU/μg DNA. The efficiency could increase to 131.5 CFU/μg DNA with the homologous extensions increased to 500 bp. pSC-MSC-flp,which hold the Flp recombinase encoding gene was transformed into E. cloacae ΔbudA-773,and resistant gene in the genome of E. cloacae ΔbudA-773 was erased after subculture. Fermentation results showed that E. cloacae ΔbudA lost the ability to synthesis acetoin and 2,3-butanediol,which indicated budA was knocked out successfully. Conclusion:An efficient gene replacement method that suit for E. cloacae was established.