Construction of adhE gene deletion mutant and compensation strains in high-alcohol producing Klebsiella pneumoniae and its effects on alcohol production
Objective To investigate the effects of adhE gene deletion on growth,alcohol dehydrogenase activi-ty,and alcohol production in high-alcohol producing Klebsiella pneumoniae(HiAlcKpn).Methods A tempera-ture-sensitive plasmid-mediated homologous recombination method was used to construct the △adhE mutant strain.The compensation plasmid was constructed by cloning the coding sequence segment of adhE into the pGEM-Teasy plasmid,then the compensation plasmid was introduced into the △adhE mutant strain.Meanwhile,the pGEM-Teasy vector was introduced into wild-type strain and △adhE mutant strain as control.The growth curves of each strain were measured in liquid LB medium,the alcohol dehydrogenase activity of each strain was detected with detection kit,and the alcohol production of each strain was detected by headspace gas chromatogra-phy(Agilent 6850)with flame ionization detection(Headspace).Results The △adhE mutant strain and com-pensation strains were successfully constructed and verified by PCR.After the deletion of adhE gene,the growth rate of HiAlc Kpn was significantly reduced under anaerobic conditions,while the growth rate of the compensa-tion strain was significantly increased(P＜0.05).At the same time,compared with the wild-type strain,the al-cohol dehydrogenase activity and the alcohol production was significantly decreased in △adhE mutant strain,while compensation with adhE restored bacterial alcohol dehydrogenase activity and alcohol production(P＜0.05).Conclusion Deletion of adhE gene slowed down the growth rate of HiAlc Kpn under anaerobic condi-tions,which significantly reduced the alcohol-producing capacity of HiAlc Kpn.
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