抗生素不同有效组分生物合成的调控机理研究

The project will focus on regulation mechanism of biosynthesis of multi-components antibiotics by investigating regulatory network of prisitinamycin biosynthesis in Streptomyces pristinaespiralis. First, all component genes of AfsKRS system from S. pristinaespiralis will be cloned, and then their sequences and structures will be analyzed. Secondly, the functions of all component genes of AfsKRS system involved in the biosynthesis of two main components of pristinamycin will be overall investigated with regulator changes, gene knockout, real-time quantitative PCR and Western blot techniques. Thirdly, transcription changes of known genes involved in pristinamycin biosynthesis were detected with real-time quantitative PCR. Fourthly, binding of AfsKRS system to the DNA fragments of pristinamycin biosynthesis gene will be investigated with gel shift assay for further study of transcription regulation. Finally, protein interactions between any two components of AfsKRS system or to other proteins were studied with yeast two-hybrid system. The project will be helpful for overall investigation of the regulation mechanism of biosynthesis of two main components of pristinamycin by AfsKRS system, and further understanding on regulatory networks of antibiotic biosynthesis. These study results will provide new strategies for yield improvement and selective control of different component biosynthesis of antibiotics by manipulating pathway regulation network.

本申请拟以普那霉素生物合成调控网络为研究对象，研究抗生素不同有效组分生物合成的基因调控机理。首先从始旋链霉菌中克隆AfsKRS调控系统的相关基因，并进行初步的序列和结构分析；利用调控系统活性调节剂、基因敲除、功能互补、荧光定量PCR和Western杂交等技术从遗传表型、转录和蛋白表达等水平研究调控系统各基因与普那霉素两类有效组分生物合成的关系；利用荧光定量PCR技术检测普那霉素两类组分生物合成基因的转录变化；利用凝胶阻滞分析技术筛选AfsKRS系统所调控的普那霉素生物合成相关DNA片段；利用酵母双杂交技术检测AfsKRS系统的各组分蛋白间及与其它蛋白间的相互作用。通过本项目的研究，明晰AfsKRS调控系统对普那霉素两类组分生物合成的调控机理，加深对多组分抗生素生物合成调控网络的认识，进而为通过遗传操作抗生素生物合成调控网络来提高其产量和进行组分控制提供新策略。

本项目研究了始旋链霉菌afsKRS系统对普那霉素不同组分生物合成的调控机理。利用DNA杂交技术从旋链霉菌基因组DNA文库中获得afsK、afsR、afsS的全长序列，生物信息学分析了afsKRS调控系统基因的结构特征；利用基因敲除技术获得了ΔafsK、ΔafsR、ΔafsS的突变体，发现afsKRS系统的不同基因对普那霉素生物合成的调控作用差别较大，afsK与普那霉素I(PI)组分的生物合成正相关，与普那霉素II(PII)组分的生物合成负相关，afsR和afsS的作用正好与afsK相反；利用SAM对ΔafsK、ΔafsR、ΔafsS突变体进行了afsKRS调控系统的激活作用研究，发现SAM通过afsKRS调控系统主要对PI组分生物合成产生激活作用，对PII组分生物合成的激活作用不明显；利用K252a对ΔafsK、ΔafsR、ΔafsS突变体进行了afsKRS调控系统的抑制作用研究，发现K252a通过afsKRS调控系统主要对PI组分生物合成发挥抑制作用，对PII组分合成的抑制作用不明显；利用荧光定量PCR、Western印迹法等技术发现afsKRS系统基因的表达水平与普那霉素高产存在相关性；荧光定量PCR还发现afsK调控PI组分生物合成的机理与snbA的表达密切相关，afsR和afsS调控PII组分生物合成的机理与snaB的表达密切相关，afsKRS调控作用还与普那霉素ptr抗性基因的转录丰度存在正相关。通过本项目的研究，初步了解afsKRS调控系统对普那霉素两类组分生物合成的调控机理，也为通过遗传操作普那霉素生物合成调控网络来提高其产量和进行组分控制提供新策略。