rDNA介导的酿酒酵母稳定表达载体的构建

Construction of rDNA-mediated stable expression vectors in Saccharomyces cerevisiae

将一段含有质粒ColE1复制起始区(ori)和氨苄青霉素抗性基因(bla)的DNA片段插入到酿酒酵母rDNA片段中部的EcoRI或HpaI位点之间,在pYES2载体的基础上构建了两个rDNA介导的酿酒酵母稳定表达载体pHBM367E或pHBM367H。将黑曲霉糖化酶基因导入载体pHBM367H,获得表达载体pHBM166。为生物安全性的考虑,pHBM166经HpaI酶切去掉2.2kb的ColE1ori和bla片段后转化酿酒酵母Y33菌株,获得不含任何抗生素抗性基因的工程菌。随后,对糖化酶基因在酿酒酵母中的表达、稳定性进行了分析,结果显示,rDNA介导的糖化酶基因在酿酒酵母中的表达呈现不同的剂量效应,挑选高表达的菌株传80代之后仍能保持其产糖化酶的稳定性。

A fragment containing ori of ColE1 and bla gene was inserted in the EcoR I or Hpa I site of the rRNA gene segment from Saccharomyces cerevisiae,respectively. Based on the expression vector pYES2 and the above two recombinant fragments,two novel rDNA-mediated stable expression vectors,designated as pHBM367E/H,were constructed. The glucoamylase gene from Aspergillus niger was inserted into the vector pHBM367H and the resultant plasmid was named pHBM166. For bio-safety consideration,the plasmid pHBM166 was linearized by Hpa I digestion to remove ColE1 ori and bla gene. Digested product was transformed into S. cerevisiae Y33. Transformants showed different expression levels of glucoamylase. Stability research showed that the selected transformant can express glucoamylase stably in S. cerevisiae for at least 80 generations.