Alcohol Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae by Suicide Plasmids Homologous Recombination:Construction and Biological Characteristics
The aim of this study was to investigate the effects of knockout of ethanol dehydrogenase gene in Enterobacter cloacae metabolic pathway on the metabolism and synthesis of acetoin,and further improve the production of acetoin.Based on the ethanol dehydrogenase(adh)gene sequence from E.cloacae SDM,a primer was designed to construct the adh gene suicide plasmid pKR6K-Δadh.Subsequently,this suicide plasmid was introduced into E.cloacae ΔbudC-ldh through heat shock method.The resulting polygene deletion strain E.cloaca Δ budC-ldh-adh was successfully constructed and subjected to fermentation analysis.The findings revealed that the recombinant strain exhibited a 20.6％increase in acetoin production intensity,a 22.6％increase in yield,and a remarkable 92.8％increase in ethanol yield compared to control strains due to successful knockout of ethanol dehydrogenase(adh)gene which in charge of microbial ethanol synthesis pathways.Furthermore,as a consequence of blocking multiple branch metabolic pathways through genetic modification,carbon flux towards succinic acid metabolic pathway increased significantly leading to an impressive 101.3％enhancement in succinic acid yield.This experiment has provided valuable insights for constructing high-yielding strains for acetoin production and establishing large-scale industrial manufacturing processes.
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