Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts
Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts
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使用从头表征的遗传部分开发链霉菌合成土霉素诱导表达系统。
DOI：
10.1021/acssynbio.6b00087
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发表时间：
2016-07-01
影响因子：
4.7
通讯作者：
Yang, Keqian
中科院分区：
文献类型：
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作者：
Wang, Weishan;Yang, Tongjian;Yang, Keqian
利用外源因子精确调控基因表达具有重要意义。为了建立理想的链霉菌诱导表达系统，我们从链霉菌中筛选了土霉素反应抑制因子OtrR、操纵子Otro和启动子otrBp这三个新的遗传部分，并对其进行了体内和体外的鉴定。OtrR对Otro有很强的亲和力(K-D=1.7×10(-10)M)，土霉素以浓度依赖的方式诱导OtrR/DNA复合体解离。在此基础上，对合成诱导表达系统PotR*进行了优化。用0.01-4MU土霉素诱导PotR*在不同种类的链霉菌中引发了GFP报告基因的广泛表达。将Potr*与广泛使用的构成启动子Erme*和kasOp*进行BenchrnarningPotr*的比对，发现当Potr*被完全诱导时，表达水平大大提高。最后，以Potr*为工具，激活和优化了静止型jadmycin生物合成基因簇在venezuelae中的表达。总之，合成的Port*为微调链霉菌的基因表达提供了一种新的多功能工具。
Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (K-D = 1.7 x 10(-10) M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01-4 mu M of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchrnarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Port* presents a new versatile tool for fine-tuning gene expression in streptomycetes.