瘤胃微生物元基因组来源的新的组成型启动子获取

New constitutive promoters screened from metagenomic library of rumen microbes

【目的】自极端环境来源的微生物的基因组中筛选新型的可用于合成生物学底盘细胞设计的启动子元件。【方法】本研究以含有绿色荧光蛋白结构基因和核糖体结合位点的探针型质粒pUC18-GFP为载体,通过构建瘤胃微生物元基因组质粒文库,从文库中快速高效筛选具有启动子功能的DNA片段。并且通过基于神经网络的启动子预测分析,获得可能的启动子区域。以绿色荧光蛋白和施氏假单胞菌Pseudomonas stutzeri来源的麦芽四糖淀粉酶作为报告基因验证所获得的新启动子片段的功能。【结果】我们从约3750个转化子中筛选到22条具有组成型启动子功能的DNA片段。这些片段与NCBI数据库中已报道的基因序列同源性较低,启动效率高低不等。我们通过启动子预测和亚克隆的方法获得两条全新的启动子片段RFa1p2(76 bp)和RFb4p(547 bp)。此新的组成型启动子可以在不添加任何诱导剂的情况下启动异源蛋白在大肠杆菌基因工程菌中高效表达。

[Objective]Screening of novel promoter elements from the genome of microorganisms of extreme environmental origin for the design of synthetic biological chassis cells.[Methods]We used a promoter-probe plasmid pUC18-GFP containing a green fluorescent protein structural gene and a ribosome bind site to construct a rumen metagenomic library.This method allows us to obtain the DNA fragments with constitutive promoter function rapidly and efficiently from this library.We obtained possible promoter regions through the neural network-based promoter prediction analysis.Then,we verified the function of the promoter initiation by using GFP and maltotetraose amylase from Pseudomonas stutzeri as the reporter.[Results]We obtained twenty-two DNA fragments functioning as constitutive promoters from about 3750 transformants.These fragments share very low sequence identities with the reported gene sequences in the NCBI database,and present different starting efficiencies.In addition,we obtained two new promoter fragments RFa1p2(76 bp)and RFb4p(547 bp)by promoter prediction and sub-cloning.These new constitutive promoters are able to express heterologous proteins efficiently in the absence of any inductor in the genetically engineered E.coli cells.