筛选地中海拟无枝酸菌无痕基因敲除菌株的简便方法

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

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。

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 dxs gene（ 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.