食甲基杆菌J1-1吡咯喹啉醌生物合成突变株的筛选和鉴定  

Screening and Identificaion of a PQQ-Deficient Mutant from Methylovorus sp. J1-1

作　　者：李彤[1,2] 景爱霞 杨亚欣 刘晓阳 汪建华[2] 刘党生 熊向华 张惟材[2] LI Tong;JING Ai-Xia;YANG Ya-Xin;LIU Xiao-Yang;WANG Jian-Hua;LIU Dang-Sheng;XIONG Xiang-Hua;ZHANG Wei-Cai(Shenyang Pharmaceutical University,Shenyang 110016,China;Beijing Institute of Biotechnology,Bejing 100071,China)

机构地区：[1]沈阳药科大学,辽宁沈阳110016 [2]军事科学院军事医学研究院,北京100071

出　　处：《生物技术通讯》2018年第2期208-213,共6页Letters in Biotechnology

摘　　要：目的:通过Tn5转座诱变筛选食甲基杆菌J1-1吡咯喹啉醌(PQQ)生物合成相关基因。方法:构建食甲基杆菌J1-1 Tn5转座突变体库,筛选PQQ合成水平差异明显的突变株,利用质粒拯救法鉴定突变基因,通过基因敲除、回补及过表达进一步研究该基因与PQQ合成的关系。结果:构建了J1-1的Tn5转座突变体库,筛选得到一株PQQ合成水平显著下降的突变株,经鉴定Tn5插入位点为mpq0056基因,该突变株在以甲醇为惟一碳源的培养基中生长速度略慢;敲除J1-1中mpq0056基因后,PQQ的合成水平下降,与Tn5诱变结果一致;回补该基因后,PQQ产量恢复到野生菌水平。结论:mpq0056基因参与了PQQ的生物合成,该基因可能编码分支酸盐裂合酶,并在PQQ生物合成中起重要作用。Objective:To screen the pyrroloquinoline quinone(PQQ)biosynthesis relative genes in Methylovorus sp.J1-1 by Tn5 transposon mutagenesis.Methods:Tn5 transposon mutants of J1-1 were obtained by kanamycin resistance screening,then the mutants whose PQQ yield changed were selected by the spectroscopy detection.The mutant genes were further identified by plasmid rescue.The role of the mutant genes in PQQ biosynthesis was determined by gene knockout,complementation and overexpression assay.Results:A mutant whose PQQ yield obviously declined was selected and the Tn5 insertion site was comfirmed in the mpq0056.In the methanol medium,the growth of the mutant was slightly declined while its PQQ synthesis level significantly decreased.A mutant of mpq0056 gene knocking-out was constructed by homologous recombination in Methylovorus sp.J1-1.The level of PQQ biosynthesis was proved to be decreased distinctly,which was consistent with the level of the Tn5 mutation above.After mpq0056 gene complementing,the yield of PQQ was restored to the normal level as the wildtype.Conclusion:It concludes that mpq0056 takes part in PQQ biosynthesis.mpq0056 gene may code chorismate lyase and play a important role in PQQ biosynthesis.

关 键 词：食甲基杆菌J1-1 吡咯喹啉醌 Tn5转座 

分 类 号：Q78[生物学—分子生物学]