摘要
利用NTG诱变从硫霉素产生菌中获得了生物合成阻断变株Y_3。通过对Y_3变株原生质体形成、再生条件及DNA转化的研究,初步建立了以变株为受体的克隆系统,以pIJ680为载体,从硫霉素产生菌S.cattleya中鸟枪克隆,获得了能使Y_3变株恢复产生硫霉素的酶基因。根据对Y_3积累的中间产物的分析,认为该酶基因可能与硫霉素生物合成过程中肽的环化作用有关。重组质粒分子大小为9.8kb左右,插入片段大小为4.5kb,分子杂交试验证明插入片段来源于硫霉素产生菌S.cattleya。
A mutant Y_3 blocked in tbienamycin biosynthetic patbway was obtained from thienamycin producing strain S. cattleya ATCC39203 by NTG treatment. Preliminary cloning system has been established on the basis of studies on the conditions for protoplast formation, regeneation, as well as DNA transformation for Y_3 mutant strain. The sbot-gun cloning was carried out from S. cattleya using pIJ680 as a vector and the Y_3 mutant as a host. A transformant No. 12 that can produce tbienamycin like substance by paper chromatography and HPLC analysis was obtained. A recombinant plasmid p6BC12 has molecular size of 9.8kb and an insert of 4.5kb could be recovered from S.lividans TK24 by transforming the DNA from transformant No. 12 into it. The intermediate accumulated by Y_3 mutant was identified as a tetrapeptide. We presume that a cyclase gene from S.cattleya was cloned according to the function of the gene product. Southern and DNA dot hybridization confirmed that the transformant No. 12 harbors the recombinant plasmid p6BC12 and the insert in p6BC12 was come from S. cattleya genome.
出处
《生物工程学报》
CAS
CSCD
北大核心
1993年第1期1-9,共9页
Chinese Journal of Biotechnology
关键词
硫霉素
生物合成酶
基因
基因克隆
Thienamycin
biosynthetase genes
blocked mutant
host system of cloning
shot-gun cloning
