Purification and Molcular Cloning of Sinefungin synthetase fom Streptomyces incarnatus NRRL8089.

新诺明是一种核苷类抗生素，其中L-欧米辛分子通过CC键连接到腺苷的5‘端。该抗生素是从Incarnatus NRRL 8089的发酵液中分离出来的，对各种真菌和锥虫有很强的抑制作用，有望作为抗疟疾药物使用。据报道，从化身变种制备的无细胞提取物在吡哆醛-5‘-磷酸存在下由L精氨酸和三磷酸腺苷产生新芬菌素，但由于产生菌的不稳定性和低表达，参与新芬菌素生产的酶尚未得到鉴定。本研究首次建立了检测菌丝菌素的DNA甲基化抑制法。然后，用紫外光照射原生质体进行诱变，得到一株高产的stra…。在获得利福平抗药性的突变体中选择了更多的In。由于在粗细胞提取液中仍未检测到菌丝菌素合成酶的活性，因此我们尝试克隆与菌丝菌素生产相关的基因。将基因组DNA部分酶切，连接到载体载体pKU110上，转化变铅青链霉菌TK24。从200多个转化子中筛选出三个转化子(#19、#20和#21)作为产生新诺明的候选菌株。转化子在2 L培养基中培养，用炭柱层析、阳离子交换柱层析和阴离子交换柱层析从发酵液中分离得到西那霉素。菌株#19比菌株#20和#21产生更多的辛那霉素。我们试图从这些产辛那霉素的转化子中分离重组质粒，但常规的分离方法没有得到重组质粒。我们通过聚合酶链式反应检测了硫链菌素抗性标记序列的存在，但相应的DNA片段没有被扩增出来。结果表明，变青链霉菌产生西诺菌素的基因已整合到变铅青链霉菌基因组DNA中。本研究发表了两篇参考文献。较少

Sinefungin is a nucleoside antibiotic, in which a molecule of L-omithine is linked to the 5'-end of adenosine through a CC bond. The antibiotic, isolated from the culture broth of Streptomyces incarnatus NRRL 8089, has a strong inhibitory effect on various fungi and trypanosome, and is expected to be useful as an anti-malaria drug as well. It has been reported that the cell-free extract prepared from a variant of incarnatus produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzymes involved in sinefungin-production have not been characterized due to the instability and low expression in the producer strain. The present study was undertaken to characterize the enzymes responsible for sinefungin production.In the present research, we have first established DNA methylse inhibition assay for detecting sinefungin. Then, the sinefungin producer strain underwent mutagenesis by ultraviolet light irradiation on the protoplast, and a high-producer stra … More in was selected among the mutants that acquired rifampicin-resistance. Because the enzyme activity of sinefungin synthetase was still not detected in the crude cell extract, we attempted to clone the genes involved in sinefungin production. The genome DNA was partially digested, ligated to a vector plasmid pKU110, and transformed in Streptomyces lividans TK24. Three transformants (#19, #20, and #21) were screened from over 200 of transformants as the candidate strain that produces sinefungin. The transformants were cultivated in 2 L of the medium, and sinefungin was isolated from the culture broth by means of charcoal column chromatography, cation-exchange column chromatography, and anion-exchange column chromatography. The strain #19 produced sinefungin more than the strains #20 and #21. We attempted to isolate the recombinant plasmids from these sinefungin-producing transformants, but the conventional isolation protocol did not yield plasmid. We examined the presence of the thiostrepton-resistance marker sequence, which is located in the vector plasmid, by polymerase chain reaction, but the corresponding DNA fragment was not amplified. It was concluded that the genes of sinefungin production from S. incarnatus were integrated to the genome DNA of S. lividans. Two references were published in the present research. Less