The effects of FNR on alcohol production of high-alcohol producing Klebsiella pneumoniae and transcriptomic analysis
Objective To investigate the effects of fnr gene deletion on alcohol production in high-alcohol produ-cing Klebsiella pneumoniae(HiAlc Kpn),and comparatively analyze wild-type Hi Alc Kpn and △fnr mutant strain by using RNA-seq technology.Methods Basing on HiAlc Kpn strain TH1,the fnr deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination.The compensation plas-mid constructed by cloning the coding sequence of fnr by PCR into plasmid pGEM-Teasy was mobilized into the△fnr mutant strain by conjugation and the pGEM-Teasy was introduced into wild-type strain and △fnr mutant strain as control.The alcohol production of wild-type strain,△fnr mutant strain and compensation strains were detected by headspace gas chromatography(Agilent 6850)with flame ionization detection(Headspace).The wild-type strain and △fnr mutant strain was analyzed using high-throughput sequencing(RNA-seq).The differ-entially expressed genes(DEGs)were screened and further analyzed using Kyoto Encyclopedia of Genes and Ge-nomes(KEGG)enrichment.Results The PCR results revealed that the △fnr mutant strain and compensation strains were successfully constructed.The alcohol production significantly decreased in △fnr mutant strain,and compensation with fnr restored bacterial alcohol production.Compared to the wild type TH1,the number of up-regulated DEGs and downregulated DEGs in △fnr mutant were 561 and 610,respectively.The KEGG enrichment analyses showed that the differentially transcribed upregulated genes in △fnr were enriched in amino acid metabo-lism,pyrimidine metabolism,and citrate cycle(TCA cycle)-related pathways.And the differentially transcribed downregulated genes in △fnr were enriched in oxidative phosphorylation,pyruvate metabolism,two-component system,biofilm formation and glycolysis/gluconeogenesis-related pathways.Conclusion Deletion of fnr gene de-creased bacterial alcohol production.And global regulator FNR can positively promote glycolytic pathway and in-hibit TCA cycle pathway in HiAlc Kpn.
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