筛选地中海拟无枝酸菌无痕基因敲除菌株的简便方法

将抗生素抗性基因作为标记筛选无痕基因敲除菌株比较费时,因而建立筛选无痕基因敲除菌株的简便方法。通过敲除茄红素生物合成途径中第一个反应的酶编码基因dxs(1-脱氧-D-木酮糖-5-磷酸合酶基因),获得白色地中海拟无枝酸菌突变菌株,以此菌株为受体菌,对S-丙二酰转移酶基因(mtf)进行无痕敲除。针对菌落本身携带颜色的地中海拟无枝酸菌(橘红色),利用茄红素合成酶基因dxs无痕敲除获得了白色菌株,在此基础上进行mtf的无痕敲除。以茄红素生物合成途径中任意一个反应的酶编码基因作为标记,很容易筛选得到无痕基因敲除的突变菌株。

Simple and Convenient Method for Screening Markerless Knock Out Strains in Amycolatopsis mediterranei

It is a time consuming to screen markerless knock out strain when an antibiotic resistance gene was used as a marker, therefore a simple and convenient method for screening markerless genetic strain was established. Adopting a knock out enzyme encoded dxsgene (1-deoxy-D-xylulose-5-phosphate synthase gene) of the first reaction in the lycopene biosynthesis pathway, a white mutant strain of Amycolatopsis mediterranei(orange red) was obtained, the strain was taken as receptor to carry out S malonyltransferase gene (mtf) markerless knock out based on this. The color of the A. mediterranei colony changed from orange red to white by knocking out dxs, and then the markerless knock out of mtf completely operated on it. Then it is easy to screen and obtain a mutant strain by using an enzyme encoded gene of any reaction in the lycopene biosynthesis pathway as a marker.