重组工程法构建德胺糖生物合成基因簇的表达载体

Construction of desosamine biosynthetic gene cluster through recombineering

德胺糖(desosamine)是次级代谢产物来源的微生物药物中的一个重要的糖基团。为构建德胺糖生物合成基因簇的异源表达体系,本研究运用重组工程技术一步法从黏粒中克隆了德胺糖的生物合成基因簇的两个转录单元,重组效率高达100%。本方法避免了常规克隆手段的烦琐、耗时、针对长DNA片段的PCR易发生碱基突变等缺点。进一步,将两个转录单元分别置于来源于天蓝色链霉菌的pacI/pactIII双向启动子的控制下并克隆至链霉菌的游离型和整合型表达载体上。本工作为探索德胺糖生物合成基因簇在异源宿主菌中表达、德胺糖与多种糖苷底物偶联以创制新化合物奠定了基础。

Desosamine is an important sugar group of microbial medicines originated from secondary metabolites. Two transcriptional units of desosamine biosynthetic gene cluster were directly cloned from the cosmid via genetic recombineering and the recombination efficiency is as high as 100%. This cloning strategy avoids the tedious, time consuming, and error-prone for long fragment PCR. The desosamine gene cluster was further put under the pacⅠ/pactⅢ bidirectional promoter from the Streptomyces coelicolor, and cloned into the streptomyces free state expression vector and integrative vector, respectively. This work sets the stage for exploring of the heterologous expression of desosamine biosynthetic gene cluster and the coupling of desosamine with various glycon substrates.