Bacillus subtilis 168菌株产surfactin改造

Reconstruction of Bacillus subtilis 168 to produce surfactin

枯草芽孢杆菌(Bacillus subtilis)168菌株基因组中含有编码surfactin合成酶系的srfA操纵子,却不产抗菌肽surfactin。运用同源重组的方法将外源的有活性的sfp基因(编码4'-磷酸泛酰巯基乙胺基转移酶)整合入B.subtilis 168中,将其改造为产surfactin的菌株。通过分析多株枯草芽孢杆菌和解淀粉液化芽孢杆菌(Bacillus amyloliquefaciens)的sfp基因,从基因的上、下游找出较为保守的区域,设计引物,从枯草芽孢杆菌Nja-9中扩增sfp基因,将获得的sfp基因与P43启动子和终止子相连,构建了sfp基因表达框,并连接于枯草整合质粒pDG1730上,最终构建了枯草芽孢杆菌整合质粒pST-1730,利用pST-1730本身的淀粉酶E基因与B.subtilis 168菌株的基因组DNA进行同源重组整合,获得了产surfactin的重组菌株B.subtilis121。本研究为surfactin合成酶基因的改造和分析奠定了基础。

The srfA operon from Bacillus subtilis 168 was previously suggested to encode putative peptide synthetases and was thought to be the surfactin operon,but B. subtilis 168 can＇t produce surfactin. The functional sfp（ encoding 4＇-phosphopantetheinyl transferase） gene was inserted to B. subtilis 168 by homologous recombination,resulting in the surfactin producing positive. Here,we amplified the funcyional sfp gene from B. subtilis Nja-9 by analysing the sfp gene with the sequence up- and down-stream from the strains belonging to B. amyloliquefaciens and B. subtilis,and ligated the functional sfp gene to P43 promoter and terminator,building up the sfp gene expression frame. The recombinant integrative vector pST-1730 was constructed and transformed to B. subtilis 168,accomplishing the integration at the amyE site of B. subtilis 168,and the surfactin-producing strain B. subtilis 121 was obtained. The study laid the foundation of the modification and analysis of the surfactin synthetase genes.