开发适用于链霉菌的新型诱导表达系统

链霉菌能够产生丰富的抗生素，是重要的工业微生物，但是，目前该菌尚没有严谨、高效的诱导表达系统，这严重制约了对其进行代谢工程与合成生物学的研究。因此，本申请项目旨在开发适用于链霉菌的新型诱导表达系统。前期，申请人根据链霉菌诱导表达系统对调控元件的具体要求，在链霉菌中筛找到能够响应土霉素等廉价小分子的MarR家族调控蛋白OtrR和CtcS及其靶启动子。所以，本研究拟首先深入解析OtrR和CtcS的调控机理，确定它们的DNA结合序列及其最佳诱导物；然后建立体内定量方法，分析这些元件在体内实现开启与关闭的相关参数及其动态过程；最后依据体内相关参数，理性组装适用于链霉菌的诱导表达系统，并利用该系统对天蓝色链霉菌放线紫红素（Act）生物合成及其前体供应途径进行“适时”、“适量”的控制，以实现Act产量的提高。本申请项目开发的诱导表达系统将为链霉菌相关研究提供方便有效的表达控制工具。

Streptomycetes are an important antibiotic producing bacteria. However, effective and convenient inducible gene expression systems that can be used for regulating the gene expression strength are currently not available in this genus. Here, novel inducible gene expression systems for streptomycetes will be developed. The systems are based on the MarR family regulatory proteins, OtrR and CtcS from streptomycetes, which were discovered to have the ability to respond to small molecules, such as oxytetracyclines. In this work, the regulatory mechanisms of OtrR and CtcS will be studied to determine their target DNA binding sequences and the kinetic parameters of ligand-protein-DNA interactions both in vitro and in vivo. Based on these information, novel inducible gene expression systems for streptomycetes will be designed. To demonstrate the applicability of the developed inducible expression systems, they will be used to coordinate the expression of actinorhodin (Act) biosynthetic cluster and the precursor supply pathway in Streptomyces coelicolor to improve Act production. These novel inducible gene expression systems will be useful tools for the genetic manipulation of streptomycetes.