大肠杆菌链长控制基因ddsA原位替换及多基因共表达.pdfVIP

食品与药品 Food and Drug 2009 年第 11卷第09期 9 大肠杆菌链长控制基因ddsA原位替换及多基因共表达 对辅酶Q10合成能力的影响 孔 璐，傅 楠，叶 江，吴海珍，张惠展* （华东理工大学生物反应器工程国家重点实验室，上海 200237） 摘 要：目的 强化大肠杆菌合成辅酶Q10 （CoQ10 ）的能力。方法 利用途径工程的基本原理，对大肠杆菌辅酶Q生物 合成途径中链长控制基因isp B进行遗传操作，并强化表达该途径中多个功能基因。结果 首次成功用来 自Gluconobacter suboxy dans 的十聚异戊二烯焦磷酸合成酶基因ddsA 原位替换大肠杆菌染色体上isp B基因，构建得到原位替换株JKL ；同时 强化表达CoQ生物合成途径的相关基因，发现在ubiCA 和ddsA 协同表达的重组菌pDCA/JKL 中CoQ 的合成能力比JKL提高了 2.3倍，CoQ10合成量提高了2.5倍。检测该重组菌中ubiA 的转录水平，发现其mRNA相对拷贝数是对照JKL 的125倍。结论 成功获得的重组大肠杆菌在降低内源性CoQ8合成能力的同时具备合成较长侧链CoQ10 的能力，且通过强化表达相关基因使 合成CoQ10 的能力得到了提高。 关键词：大肠杆菌；ddsA ；基因替换；共表达；辅酶Q10 ；qRT-PCR 中图分类号：Q78 文献标识码：A 文章编号：1672-979X （2009 ）09-0009-06 Influence of Gene Substitution with Gluconobacter Suboxydans ddsA and Multi-gene Co- expression on Production of Ubiquinone-10 in Escherichia coli * KONG Lu, FU Nan, YE Jiang, WU Hai-zhen, ZHANG Hui-zhan (State Key Lab of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China) Abstract: Objective To overproduce ubiquinone- 10 in Escherichia coli. Methods By the principle of pathway engineering, a series of gene manipulations of isp B gene which encodes octaprenyl pyrophosphate synthase in the ubiquinone biosynthesis of E. coli JM83 were taken. Also several genes were overexpressed, which played a central role in this metabolic pathway. Results The isp B gene of Escherichia coli JM83, which encodes octaprenyl pyrophosphate synthase in the ubiquinone biosynthesis, was first successfully replaced by the ddsA gene of Gluconobacter suboxy dans which encodes decaprenyl pyrophosphate synthase. This substitution strain was designated as JKL. The several genes which played a c