Gene amplification induced by a tunicamycin-resistant mutation in Bacillus subtilis and its application to protein production.

Research Project

Project/Area Number	60470129
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	発酵・醸造
Research Institution	Faculty of Agriculture, The University of Tokyo
Principal Investigator	YAMASAKI Makari Department of Agricultural Chemistry, The University of Tokyo, 農学部, 教授 (60011889)
Co-Investigator(Kenkyū-buntansha)	YODA Koji Department of Agricultural Chemistry, The University of Tokyo, 農学部, 助手 (20143406)
Project Period (FY)	1985 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥5,800,000 (Direct Cost: ¥5,800,000) Fiscal Year 1986: ¥1,400,000 (Direct Cost: ¥1,400,000) Fiscal Year 1985: ¥4,400,000 (Direct Cost: ¥4,400,000)
Keywords	Bacillus subtilis / Tunicamycin-resistance / Gene amplification / <alpha> -Amylase / シキミ酸キナーゼ
Research Abstract	The aim of this study is to induce a designed gene ampliciation on the chromosome of Bacillus subtilis and its application to protein production. We have already elucidated the essential structure of a transforming DNA which can induce gene amplifcation in B. subtilis. The juction point structure of the repeating unit is essential for the active transformation. If so, we can construct in in vitro a transforming DNA by ligating two independent DNA fragments to form a juction point of the designed repeating unit. From the chromosomal DNA of B. subtilis, we cloned a 5.1 kb EcoRI fragment which is located at the upstream of amyE and also a 0.8 kb Hind <III> -ClaI fgrament which is located near aroI. The two DNA fragments were ligated in in vitro to construct a juction point of the aimed repeating unit (22 kb). By competence transformation with 10 ug of the constructed DNA, 10 tunicamycin-resistant transformants were obtained. Among them four strains showed hyper productivity of <alpha> -amylase. We selected two of the hyper strains for the further analyses. Restriction endonuclease-digested chromosomal DNAs were subjected to an agarose gel electrophoresis and Southern hybridization with several appropriate probes. All the data confirmed the occurrence of the designed gene amplification having a 22 kb repeating unit. <alpha> -Amylase hyper productivity and tunicamycin-resistance were brought about by the simulteneous amplification of amyE and tmrB genes. Shikimate kinase activity was also enhanced by 4 folds in accordance with the amplification of aroI.