食甲基菌吡咯喹啉醌生物合成相关基因的筛选及表型鉴定  

Screening and Phenotypic Identification of Gene Related to Biosynthesis of Pyrroloquinoline Quinone in Methylovorus sp.J1-1

作　　者：薄明井 寇航 陈静 张惟材 葛欣[1] 熊向华 BO Ming-Jing;KOU Hang;CHEN Jing;ZHANG Wei-Cai;GE Xin;XIONG Xiang-Hua(School of Life Sciences,Hebei University,Baoding 071000;Beijing Institute of Biotechnology,Beijing 100071,China)

机构地区：[1]河北大学生命科学学院,河北保定071000 [2]军事医学研究院生物工程研究所,北京100071

出　　处：《生物技术通讯》2020年第6期653-658,共6页Letters in Biotechnology

基　　金：国家科技重大专项(2018X09J18109-003)。

摘　　要：目的:通过Tn5转座诱变筛选食甲基菌中吡咯喹啉醌(PQQ)生物合成相关基因。方法:通过Tn5转座诱变食甲基菌J1-1,筛选PQQ合成水平变化明显的突变株,利用质粒拯救法鉴定突变位点,进而敲除对应基因验证其与PQQ合成的关系,再检测野生菌及敲除菌的NADH和NAD+含量。结果:构建了库容量为1.0×105的Tn5转座突变体库,筛选得到1株PQQ合成水平显著下降的突变株5-179,经鉴定Tn5转座子插入染色体第705454位,位于mpq-0708基因内部(705337~705933),该基因编码NADH醌氧化还原酶亚基C;通过同源重组敲除J1-1中mpq-0708基因,敲除菌在高产培养基中PQQ合成水平显著下降,仅为野生菌的3.15%,与5-179突变株表型相符;野生菌及敲除菌NADH与NAD+测定结果表明,NADH/NAD+比值代表的菌体能量代谢状态与PQQ生物合成及菌体生长显著关联。结论:mpq-0708基因可能通过能量代谢参与PQQ生物合成。Objective:To identify pyrroloquinoline quinone(PQQ)biosynthesis genes in Methylovorus sp.J1-1 by Tn5 transposon mutagenesis.Methods:Tn5 transposon mutants from Methylovorus sp.J1-1 were isolated and screened,the mutant genes were further identified by plasmid rescue.Then,the corresponding genes were knocked out to verify their relationship with PQQ biosynthesis.At last,the NADH and NAD+content of Methylovorus sp.J1-1 and the mutant were determined and their ratio was calculated to analyze the cell state.Results:A mutant 5-179 with obvious PQQ yield change was obtained by Tn5 mutagenesis.Tn5 was identified as being inserted in⁃to the open reading frame of the mpq-0708 gene at 705454 loci,which encodes NADH quinone oxidoreductase subunit C(ORF:705337~705933).After knocking out the mpq-0708 gene by homologous recombination,PQQ yield of the mutant in HM medium was only 3.15%of that of the wild strain,which perfectly coincided with 5-179.The NADH/NAD+ratio showed that cell energy metabolism level is greatly related to PQQ biosynthesis and bacterial growth.Conclusion:The mpq-0708 gene may be involved in PQQ biosynthesis through energy metabo⁃lism.

关 键 词：Tn5转座 吡咯喹啉醌 NADH/NAD+ 能量代谢 食甲基菌 

分 类 号：Q78[生物学—分子生物学]