Hasegawa M, Dairi T, Ohta T, Hashimoto E
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
J Bacteriol. 1991 Nov;173(21):7004-11. doi: 10.1128/jb.173.21.7004-7011.1991.
A highly efficient gene-cloning system for Micromonospora olivasterospora, a producer of the antibiotic fortimicin A (astromicin), suited to shotgun cloning has been developed. The system is supported by two new advancements accomplished in this study. One is the construction of novel plasmid vectors pMO116, pMO126, pMO133, pMO136, and pMO217, all consisting of replicons from newly found Micromonospora plasmids and selectable markers cloned from a neomycin-producing Micromonospora strain. The other advancement is the establishment of a new protocol for bacterial protoplasting in which some kinds of sugar alcohols are added in precultures. Such sugar alcohols were found to sensitize a wide taxonomical range of bacteria to lysozyme. The system is reproducible and reliable and has a high efficiency of more than 10(6) CFU/micrograms of DNA.
已开发出一种适用于鸟枪法克隆的高效基因克隆系统，用于生产抗生素福提霉素A（阿司米星）的橄榄小单孢菌。该系统得到了本研究中完成的两项新进展的支持。一是构建了新型质粒载体pMO116、pMO126、pMO133、pMO136和pMO217，它们均由新发现的小单孢菌质粒的复制子和从产生新霉素的小单孢菌菌株克隆的选择标记组成。另一项进展是建立了一种新的细菌原生质体制备方案，其中在预培养物中添加了某些种类的糖醇。发现这些糖醇能使广泛分类范围的细菌对溶菌酶敏感。该系统可重复且可靠，DNA效率超过10(6) CFU/微克，具有较高的效率。
