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

Research Project

Project/Area Number	59860009
Research Category	Grant-in-Aid for Developmental Scientific Research
Allocation Type	Single-year Grants
Research Field	発酵・醸造
Research Institution	University of Tokyo
Principal Investigator	YANO Keiji Faculty of Agriculture, University of Tokyo, 農学部, 教授 (10011842)
Co-Investigator(Kenkyū-buntansha)	江井仁味の素株式会社, 中央研究所, 主任研究員 FUKUDA Masao Faculty of Agriculture, University of Tokyo, 農学部, 助手 (20134512) TAKAGI Masamichi Faculty of Agriculture, University of Tokyo, 農学部, 助教授 (50018339) ENUI Hitoshi Ajinomoto Co., Inc.
Project Period (FY)	1984 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥6,500,000 (Direct Cost: ¥6,500,000) Fiscal Year 1986: ¥700,000 (Direct Cost: ¥700,000) Fiscal Year 1985: ¥1,400,000 (Direct Cost: ¥1,400,000) Fiscal Year 1984: ¥4,400,000 (Direct Cost: ¥4,400,000)
Keywords	Gram-Negative Bacteria / Broad Host Range Vector / Gene Cloning System / Enzyme Production / Alkylbenzene / Oxidative Enzymes / Extracellular Protease / シュードモナス / 遺伝子クローニング / 酵素生産法
Research Abstract	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