一种新的链霉菌表达载体启动子论文.docVIP

　　一种新的链霉菌表达载体启动子论文 吴杭 张部昌 查向东 孔小卫 马清钧 【摘要】 eryA基因直接控制着红霉素母环6-脱氧-红霉内酯B的合成，在红霉素生物合成过程中具有重要作用。本文克隆了eryA基因的启动子PeryA，以绿色荧光蛋白基因为报告基因，构建了大肠埃希菌-糖多孢红霉菌穿梭型质粒。PEG介导原生质体转化法将穿梭型质粒分别转入糖多孢红霉菌A226与变铅青链霉菌JT46，荧光显微镜检测发现，此启动子在两菌株中都具有功能。随后，以变铅青链霉菌JT46为宿主，对PeryA启动子区域进行了深入研究.freelportant role in the biosynthesis of erythromycin macrolactone ring (6-deoxy-erythronolide B, 6-dEB). In this paper, an Escherichia coli-Saccharopolyspora erythraea shuttle vector containing the eryA promoter region id ed into Sac.erythraea A226 and Streptomyces lividans JT46 by PEG mediation, respectively. Fluorescence microscopy confirmed that the EGFP oter region oter and the 41-bp promoter DNA fragment only containing predicted -10 region yces. Site-directed mutant demonstrated that the predicted -10 region oter. Thus, the 41-bp promoter segment can function as an effective promoter of Streptomyces expression vector, oters hitherto found in Streptomyces and is useful for constructing neyces expression vectors. KEY aterials and methods 1.1 Strains, plasmids and culture conditions Sac.erythraea A226 and S.lividans JT46 Prof. edicinal Biotechnology, Chinese Academy of Medical Sciences. Sac.erythraea A226 ants. Escherichia coli DH5α aintained according to the standard method［17］. The details of Sac.erythraea and S.lividans culture anipulation and cloning. Plasmids pEGFP-N1 (Clontech, U.S.A.), pUEKML［18］ and pCS) and fd transcription terminator oved from pUEKML e sites of pUP, yielding pUPKML. PCR ed using plasmid pEGFP-N1 as a template, and the PCR product pUC-EGFP e site of p-EGFP［18］. Therefore, ants as positive controls. The plasmid pUPironov V A, Sergienko O V, Nastasiak I N, et al. Biogenesis and regulation of biosynthesis of erythromycins in Saccharopolyspora erythraea ［J］. Appl Biochem Microbiol,2004,40(6):613 ［2］ Fernandez-Moreno M A, Martinez E, Boto L, et al. Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase