Development of the method for the useful-enzyme production using broad host range vectors

虽然具有大肠杆菌K-12的宿主载体系统已被证明对于基因克隆具有巨大价值，但它们在许多革兰氏阴性细菌中不可用。在这项研究中，我们的第一个目标是建立一个基因克隆系统，可在许多革兰氏阴性菌。我们以高拷贝、广宿主范围质粒RSF 1010为基础，构建了克隆载体pMFY 31（<Ap ^r>，<Cm ^r>，<Tc^r>，13.2kb）和pMFY 40（<Ap^r>，<Tc^r>，11.6kb）。这些载体已被有效地引入到恶臭假单胞菌菌株，不仅通过质粒DNA转化，而且通过接合共转移，并保持稳定在该菌株。我们的基因克隆系统包括以下三个步骤。(1)利用广宿主范围粘粒载体在大肠杆菌中构建基因文库。(2)将基因文库接合共转移到靶菌株中并选择阳性克隆。(3)重组子在大肠杆菌中的亚克隆、DNA分析及表型鉴定 关于我们 目标菌株的特征。我们对每一步进行了研究，建立了完整的系统，并在此系统中克隆了一些革兰氏阴性菌的基因，以期提高有价值的酶的产量。我们已经克隆了恶臭假单胞菌的alk基因，该基因促进正烷烃和烷基苯的氧化。然后，我们将alkBAC操纵子片段亚克隆到新构建的载体pMFY 40上，并从恶臭假单胞菌中的互补实验中确定基因区域。为分离荧光假单胞菌胞外蛋白酶基因，构建了荧光假单胞菌胞外蛋白酶基因文库，并通过接合转移法将其转入恶臭假单胞菌，但未获得阳性克隆。在此基础上，我们设计了利用人工合成的寡核苷酸探针进行基因克隆的实验。我们已经进行了蛋白酶的部分纯化，并可以鉴定蛋白质。在这些实验中，我们的基因克隆系统的可用性和我们的载体的有用性被认为是认证，我们已经获得了开发有用的酶生产方法的优秀工具。少

While host-vector systems with Esherichia coli K-12 have proven of immense value for the cloning of genes, they are not available in many Gram-negative bacteria. In this study we first aim to establish a gene cloning system that is available in many Gram-negative bacteria. We newly constructed cloning vectors, pMFY31( <Ap^r> , <Cm^r> , <Tc^r> , 13.2kb) and pMFY40( <Ap^r> , <Tc^r> , 11.6kb) based on the high-copy-number, broad-host-range plasmid RSF1010. These vectors has been efficiently introduced into a Pseudomonas putida strain not only by plasmid-DNA transformation but also by conjugal co-transfer and were maintained stably in this strain. Our gene cloning system includes three steps as follows. (1) Construction of a gene library in E.coli using a broad-host-range cosmid vector. (2) Conjugal co-transfer of a gene library into the targeted strain and selection of the positive clones. (3) Subcloning and DNA analysis of the recombinant in E.coli, and determination of their phenotypic … More characteristics in the targeted strain. We have examined the each steps and built up whole the system.Next, we aim to clone genes of some Gram-negative bacteria in this system with a special view to increasing the production of the enzymes of great value. We have cloned the alk genes of P.putida that promotes the oxidation of both n-alkane and alkylbenzene. Then, we subcloned the alkBAC operon fragments onto the newly constructed vcctor pMFY40 and determine the gene region from complementation experiments in P.putida. To isolate the extracellular protease gene of P.fluorescens, the gene library was constructed and transfered into the P.putida strain by conjugation, however no positive clone was isolated. Then we have planed the gene cloning experiment using synthetic oligo-nucleotide probe. We have carried out partial purification of the protease and could identified the protein.In these experiments the availability of our gene cloning system and the usefulness of our vectors were thought to be certified and we have acquired excellent tools for the development of the method for the useful-enzyme production. Less