Preparation and characterization of sinefungin synthetase

新诺明是一种核苷类抗生素，其中L-蛋氨酸分子通过C-C键连接到腺苷的5‘端。该抗生素是从化身为NRRL8089的链霉菌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，有望用作抗疟疾药物。据报道，化身链球菌变种的无细胞提取液在吡哆醛-5‘-磷酸存在下由L-精氨酸和三磷酸腺苷产生新芬菌素，但参与生产新芬菌素的酶(S)尚未得到鉴定，这表明产生菌中负责酶的不稳定和低表达。本研究在pKU110穿梭载体的基础上，构建了一个新的pTRA502表达载体。本研究旨在鉴定与新诺菌素生物合成相关的基因。用pKU110的新霉素抗性基因替换硫链菌素抗性基因，构建了pTRA502的原核表达载体。用Sau3AI限制性内切酶对基因组DNA进行限制性内切酶消化，得到约5-6kb的遗传片段。通过限制性内切酶浓度、酶浓度和培养时间确定了最适培养条件。在提取DNA的过程中，存在DNA收集率低的问题。玻璃珠试剂盒的使用提高了玻璃微珠的收集量和纯度。目前正在利用pTRA502载体进行枪击克隆。用毛细管电泳法对转化子进行了产辛那霉素能力的检测。

Sinefungin is a nucleoside antibiotic, in which a molecule of L-methionine is linked to the 5'-end of adenosine through a C-C bond. The antibiotic, isolated from the culture broth of Streptomyces incarnates NRRL8089, 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 S. incarnates produced sinefungin from L-arginine and ATP in the presence of pyridoxal-5'-phosphate, but the enzyme(s) involved in sinefungin-production have not been characterized clue to the instability and low expression of the responsible enzymes in the producer strain. In the present study we have constructed a new expression-vector of pTRA502, which derives from pKU110 shuttle vector. The present study was undertaken to identify genes involved in the sinefungin biosynthesis. We have constructed an original cloning vector of pTRA502 by replacing thiostrepton resistant gene with that of neomycin resistant gene of pKU110. Limited digestion of genome DNA was carried out with Sau3AI restrictions enzyme to obtain genetic fragments of about 5-6 kb. Optimal conditions were examined by restriction enzyme concentrations, enzyme concentration and incubation time. There was a problem that the collection of DNA was low in the process to extract DNA. The amount of collection and purity was improved by using the kit of glass beads. Shot-gun cloning is now being carried out with pTRA502 vector. The transformants were examined for the sinefungin producing ability by a capillary electrophoresis.