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

虽然大肠杆菌K-12的宿主-载体系统已被证明对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，我们首先旨在建立一个可用于许多革兰氏阴性菌的基因克隆系统。基于高拷贝数、宽宿主范围的质粒RSF1010，构建了pMFY31（<Ap^r>, <Cm^r>, <Tc^r>, 13.2kb）和pMFY40（<Ap^r>, <Tc^r>, 11.6kb）克隆载体。这些载体不仅可以通过质粒dna转化，还可以通过偶联共转移有效地导入到恶臭假单胞菌中，并在该菌株中保持稳定。我们的基因克隆系统包括以下三个步骤。(1)利用广宿主cosmid载体构建大肠杆菌基因文库。(2)将基因文库偶联共转移到目标菌株中，筛选阳性克隆。(3)重组菌株在大肠杆菌中的亚克隆和DNA分析，并测定其表型特征。我们检查了每一个步骤，并建立了整个系统。下一步，我们的目标是在该系统中克隆一些革兰氏阴性菌的基因，以增加有价值的酶的产量。我们克隆了一种促进正构烷烃和烷基苯氧化的恶臭P.putida的alk基因。然后，我们将alkBAC操纵子片段亚克隆到新构建的载体pMFY40上，并通过在p.p putida中的互补实验确定了基因区域。为了分离胞外蛋白酶基因，构建了荧光p.f urcens基因文库，并通过偶联法将其转移到p.p putida菌株中，但未分离到阳性克隆。然后计划了利用人工合成的寡核苷酸探针进行基因克隆实验。我们对该蛋白酶进行了部分纯化，并对其进行了鉴定。在这些实验中，我们的基因克隆系统的可用性和载体的有用性被认为是经过认证的，我们已经获得了开发有用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