硫霉素环化酶基因在变铅青链霉菌TK24中的表达及基因定位

将含有硫霉素环化酶基因的重组质粒p6BCl2转化变铅青链霉菌(Streptomyceslividans)TK24，含有p6BCl2的转化子细胞抽提液分别与琉霉素生物合成阻断变株Y，发酵液以及纯化的Y。中间产物经过体外共培养可产生活性物质．化学分析表明与Y，发酵液混合后产生的是硫霉素，与纯化的Y。中间产物混合产生的是一种不稳定的活性物质。说明硫霉素环化酶基因在S.lividans TK24中得到了表达，其产物以Y。中间产物为底物并弥补了Y，中的缺陷。对p6Bcl2中4．5kb外源片段进行了限制酶酶切分析，建立了酶切图谱．利用含硫霉素环化酶基因的S．Lividans TK24转化子体外转化Y，的应用体系，将硫霉素环化酶基因定位在0．9kb Hinc I—Pst I片段上，并证明了硫霉紊环化酶的活性与IPNS同源片段无关。以上实验为进一步研究琉霉素环化酶基因的结构打下了基础。

Gene Localization and Expression of Thienamycin Cyclase Gene in Streptomyces lividans TK24

The transformants of 5. lividans TK24 were obtained by transforming of the recombinant plasmid p6BCl2 harboring the thienamycin cyclase gene into it. An antibacterial substance could be detected by conversion of fermentation broth of Y3 block mutant and purified Y3 mutant intermediate with cell-free extract of S. lividans TK24 trans-formant, respectively. Paper chromatographic analysis showed that the cell-free extract could convert Y3 fermentation broth to thienamycin and purified Y3 intermediate product to an antibacterial substance in vitro. This indicated that the thienamycin cyclase gene could be expressed in S. lividans TK24 and the cyclase gene could complement the deficiency in Y3 block mutant. Restriction analysis of p6BC12 was carried out and the restriction map was constructed. The thienamycin cyclase gene was localized on a Pst I -Mine II 0. 9kb fragment from bioconvertion result. The 1. 0kb IPNS homologous DNA fragment in plasmid p6BC12 of cyclase gene was excluded from the cyclase activity.