Weber G, Leitner E
Biochemie Ges.m.b.H., Kufstein-Schaftenau, Austria.
Curr Genet. 1994 Nov-Dec;26(5-6):461-7. doi: 10.1007/BF00309935.
Cyclosporin A is a potent and clinically-important immunosuppressive drug (SandimmunR). It is produced by the fungus Tolypocladium niveum. A transformation system for T. niveum ATCC34921 based on hygromycin selection was established. In order to obtain a T. niveum promoter, the cyclophilin gene was isolated using the Neurospora crassa gene as probe. A plasmid vector was constructed in which the promoter region of the T. niveum cyclophilin gene was fused to a bacterial hygromycin phosphotransferase gene. Protoplasts were transformed with this plasmid and hygromycin-resistant transformants were isolated. Using this transformation system, mutants of T. niveum with disrupted versions of the cyclosporin synthetase gene (simA) were engineered by DNA-mediated transformation. Disruption of the gene resulted in loss of the ability to produce cyclosporins.
环孢菌素A是一种强效且具有临床重要性的免疫抑制药物（山地明®）。它由真菌雪白丝枝霉产生。基于潮霉素筛选建立了雪白丝枝霉ATCC34921的转化系统。为了获得雪白丝枝霉启动子，以粗糙脉孢菌基因作为探针分离亲环素基因。构建了一个质粒载体，其中雪白丝枝霉亲环素基因的启动子区域与细菌潮霉素磷酸转移酶基因融合。用该质粒转化原生质体并分离出潮霉素抗性转化体。利用这个转化系统，通过DNA介导的转化构建了具有环孢菌素合成酶基因（simA）破坏版本的雪白丝枝霉突变体。该基因的破坏导致产生环孢菌素的能力丧失。
