通过突变甲醇脱氢酶启动子提高甲基营养菌吡咯喹啉醌产量  

Increasing Pyrroloquinoline Quinone Yield of Methylovorus sp. by Methanol Dehydrogenase Promoter Mutation

作　　者：孙晓宇 薄明井 杨亚欣 张惟材 葛欣[1] 熊向华 SUN Xiao-Yu;BO Ming-Jing;YANG Ya-Xin;ZHANG Wei-Cai;GE Xin;XIONG Xiang-Hua(Hebei University Academy of Life Science,Baoding 071000,China;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]军事科学院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》2019年第5期609-613,692,共6页Letters in Biotechnology

基　　金：国家科技重大专项（2018X09J18109-003）

摘　　要：目的:考察甲基营养菌J1-1甲醇脱氢酶启动子活性与吡咯喹啉醌(PQQ)产量的相关性。方法:通过定点突变甲醇脱氢酶P0771启动子,利用lacZ作为报告基因检测突变启动子的转录水平,筛选出活性下降的突变启动子在J1-1中替换天然启动子获得突变菌株,检测突变菌株甲醇脱氢酶表达、酶活以及菌体生长和PQQ产量。结果:定点突变了7个P0771启动子,利用lacZ作为报告基因筛选出2个启动活性较天然启动子下降的突变启动子P0771-1、P0771-5,替换J1-1中天然启动子获得的突变菌株甲醇脱氢酶酶活都低于J1-1,在增菌培养基中其PQQ的合成水平分别提高约9.76%、11.6%。结论:通过启动子突变,降低甲醇脱氢酶启动子活性可以提高J1-1的PQQ产量。Objective:The correlation between methanol dehydrogenase promoter activity and pyrroloquinoline quinone(PQQ)yield of methylotrophic bacteria J1-1 was investigated.Methods:The mutants of methanol dehydrogenase promoter P0771 were constructed by site-directed mutagenesis,and lacZ was used as a reporter gene to detect the transcription level of the mutant promoter.The low-activity mutant promoter was selected to replace the natural promoter in J1-1 to obtain the mutant strain.The methanol dehydrogenase expression,enzyme activity,and bacterial growth and PQQ production were examined in the mutant strain.Results:Seven P0771 mutant promoters were obtained by site-directed mutagenesis,and thereinto two promoters P0771-1 and P0771-5 with lower transcriptional levels than the native promoter were screened by using lacZ as a reporter gene to replace the natural promoter in J1-1.The enzyme activity of obtained mutant strain was lower than J1-1,and the yield of PQQ in the enrichment medium was increased by about 9.76%and 11.6%,respectively.Conclusion:By reducing the methanol dehydrogenase promoter activity with promoter mutations,the PQQ yield of J1-1 can be increased.

关 键 词：甲基营养菌 定点突变 甲醇脱氢酶 吡咯喹啉醌 

分 类 号：Q78[生物学—分子生物学]