Transformation of Streptomyces erythraeus.
Transformation of Streptomyces erythraeus.
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DOI：
10.7164/antibiotics.39.1304
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发表时间：
1986-09
期刊：
影响因子：
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通讯作者：
H. Yamamoto;K. Maurer;C. Hutchinson
中科院分区：
文献类型：
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作者：
H. Yamamoto;K. Maurer;C. Hutchinson
通过使用0.2mM Ca 2+、5 mM Mg 2+和10%DMSO的方案，用六种不同的质粒DNA载体有效地转化Streptomycesisraeus菌株，以增强原生质体在-80 ℃下储存的稳定性和它们在30 ℃下的可转化性。大多数载体的初级硫链丝菌素抗性（Thior）转化体即使在选择性生长时也不稳定。这种不稳定性似乎不是由于与土著S.螺旋体质粒。相反，稳定转化不是质粒或宿主突变的结果。因此，转化不稳定性或质粒拷贝数阻止了补充eryD突变的DNA的成功鸟枪式克隆，这阻断了红霉素A的生物合成，因为只有没有插入片段的质粒DNA可以从Thior Ery+克隆中分离出来。
Streptomyces erythraeus strains were transformed efficiently with six different plasmid DNA vectors by a protocol that uses 0.2 mM Ca2+, 5 mM Mg2+ and 10% DMSO to enhance the stability of protoplasts to storage at -80 degrees C and their transformability at 30 degrees C. The primary thiostrepton-resistant (Thior) transformants for most vectors were unstable even when grown selectively. This instability did not appear to be due to incompatibility with indigenous S. erythraeus plasmids. Conversely, stable transformation was not the result of plasmid or host mutations. Transformation instability or plasmid copy number thus prevented successful shotgun-cloning of DNA that complemented the eryD mutation, which blocks the biosynthesis of erythromycin A, because only plasmid DNA without an insert could be isolated from a Thior Ery+ clone.