Dairi T, Ohta T, Hashimoto E, Hasegawa M
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
Mol Gen Genet. 1992 Mar;232(2):262-70. doi: 10.1007/BF00280005.
We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employes the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pMO116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.
我们使用一种最近开发的自克隆系统克隆了负责抗生素福提霉素A（FTM A）生物合成的七个基因，该系统采用质粒载体pMO116用于橄榄色小单孢菌。通过鸟枪法克隆分离出五个嵌合质粒，这些质粒恢复了在不同生物合成步骤受阻的橄榄色小单孢菌突变体中FTM A的产生。使用其他不产生产物的突变体进行二次转化表明，这些质粒上还包含另外两个FTM A生物合成基因，并且至少有四个基因是成簇的。有趣的是，AN38-1是一个不产生产物的突变体，在FTM A前体的脱羟基化方面存在缺陷，在构建质粒载体pMO116的过程中，从产生新霉素的菌株（小单孢菌属FTM A不产生产物菌株）克隆的含有新霉素抗性基因的DNA片段对其进行了互补。这些结果清楚地表明，这种在小单孢菌中的新型基因克隆系统具有实际用途。
