FNR影响高产乙醇肺炎克雷伯菌产乙醇能力及转录组分析

The effects of FNR on alcohol production of high-alcohol producing Klebsiella pneumoniae and transcriptomic analysis

目的探究fnr基因缺失对高产乙醇肺炎克雷伯菌产乙醇能力的影响,并对野生型菌株与Δfnr缺失菌株进行转录组测序分析。方法以高产乙醇肺炎克雷伯菌株TH1为背景,利用温敏型质粒介导的同源重组技术构建fnr基因的缺失菌株。PCR扩增并克隆fnr基因于pGEM-Teasy表达载体获得回补质粒,导入Δfnr缺失菌株得到回补株,并将pGEM-Teasy空质粒导入野生株及缺失株作为空载对照。通过顶空法检测基因fnr缺失对高产乙醇肺炎克雷伯菌株乙醇产量的影响。并对野生型菌株和Δfnr缺失菌株进行转录组测序,筛选差异表达基因并进行京都基因与基因组百科全书(KEGG)富集分析。结果通过PCR技术明确fnr基因缺失与回补菌株构建成功。乙醇含量测定结果显示基因fnr缺失后,高产乙醇肺炎克雷伯菌株的乙醇产量显著降低,且回补菌株的乙醇产量显著升高。转录组测序结果表明基因fnr缺失后561个基因表达上调,610个基因表达下调。KEGG富集分析结果表明,上调基因主要富集在包括氨基酸代谢、嘧啶代谢、三羧酸循环等通路,下调基因中富集到了包括氧化磷酸化、丙酮酸代谢、双组份系统、磷酸转移酶系统(PTS)、生物被膜形成、糖酵解/糖异生等通路。结论fnr基因的缺失显著降低了高产乙醇肺炎克雷伯菌株的乙醇产量,且全局性调控因子FNR能够正向促进糖酵解通路,抑制三羧酸循环通路。

Objective To investigate the effects of fnr gene deletion on alcohol production in high-alcohol producing Klebsiella pneumoniae(HiAlc Kpn),and comparatively analyze wild-type HiAlc Kpn andΔfnr 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 plasmid constructed by cloning the coding sequence of fnr by PCR into plasmid pGEM-Teasy was mobilized into theΔfnr mutant strain by conjugation and the pGEM-Teasy was introduced into wild-type strain andΔfnr mutant strain as control.The alcohol production of wild-type strain,Δfnr mutant strain and compensation strains were detected by headspace gas chromatography(Agilent 6850)with flame ionization detection(Headspace).The wild-type strain andΔfnr mutant strain was analyzed using high-throughput sequencing(RNA-seq).The differentially expressed genes(DEGs)were screened and further analyzed using Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment.Results The PCR results revealed that theΔfnr mutant strain and compensation strains were successfully constructed.The alcohol production significantly decreased inΔfnr mutant strain,and compensation with fnr restored bacterial alcohol production.Compared to the wild type TH1,the number of upregulated DEGs and downregulated DEGs inΔfnr mutant were 561 and 610,respectively.The KEGG enrichment analyses showed that the differentially transcribed upregulated genes inΔfnr were enriched in amino acid metabolism,pyrimidine metabolism,and citrate cycle(TCA cycle)-related pathways.And the differentially transcribed downregulated genes inΔfnr were enriched in oxidative phosphorylation,pyruvate metabolism,two-component system,biofilm formation and glycolysis/gluconeogenesis-related pathways.Conclusion Deletion of fnr gene decreased bacterial alcohol production.And global regulator FNR can positively promote glycolytic pathway and inhibit TCA cycle pathway in HiAlc Kpn.