基于TALE-TF技术调控纤维堆囊菌埃博霉素高效生物合成及其机制研究

Epothilones produced by Sorangium cellulosum show broad-spectrum and high-potency antitumor acitivity and is difficult to generate drug resistance. However,the deficiency in the genetic manipulation methods for S. cellulosum and insufficiency of research on the regulation mechanism of epothilones biosynthesis limit high-efficiency biosynthesis of epothilones in S. cellulosum. TALE-TF technique has been broadly applied in the enhancement of target gene transcription activity. Based on the epothilone-producing S. cellulosum So ce M4 isolated in our lab, the establishment of genetic manipulation system and prelimiary identification of epothilones gene cluster promoter of this strain were implemented, this project aims to enhance the transcription activity of epothilone-producing related genes employing the TALE-TF technique, thereby achieving high-efficiency biosynthesis of epothilones in S. cellulosum So ce M4, to investigate the synergic effect of TALE-TF elements by the comparison of epothilones yield, target gene transcription activity and the comparison of abundance of target protein in original and recombinant strain by 2D electrophoresis. Chromatin chromatin immunoprecipitation and biomolecular interaction analysis will be employed to investigate the interaction of TALE-TF element and promoter sequence of epothilone gene cluster of S. cellulosum So ce M4 in vivo and in vitro. This research will reveal the regulation mechanism of epothilones biosynthesis in S.cellulosum, thereby laying a theoretical foundation for the high-efficiency biosynthensis of epothilones.

纤维堆囊菌的次级代谢产物埃博霉素具有广谱高效的抗肿瘤活性且不易产生耐药性，但纤维堆囊菌遗传操作体系比较匮乏，且对埃博霉素生物合成调控机理尚不清楚，因此很难从分子水平显著提高纤维堆囊菌中埃博霉素合成效率。本课题组在前期工作中自主分离得到了产埃博霉素纤维堆囊菌菌株So ce M4，建立了该菌株遗传操作体系，并对其埃博霉素基因簇启动子进行了初步鉴定。TALE-TF技术在增强目的基因转录活性方面有广泛应用。本项目拟利用TALE-TF技术增强菌株So ce M4埃博霉素生物合成基因簇的转录活性，实现埃博霉素的高效生物合成，通过目的基因的转录活性、表达水平及埃博霉素产量的分析，研究TALE-TF元件的协同效应，并应用ChIP技术和BIA技术分别研究TALE-TF元件与启动子序列在纤维堆囊菌中及体外的相互作用，揭示TALE-TF元件调控纤维堆囊菌埃博霉素生物合成的机制，为埃博霉素的高效生物合成奠定基础。

埃博霉素是一种具有广谱抗肿瘤活性的大环内酯类化合物，其衍生物已经批准用于临床治疗乳腺癌。但其较低的产量限制了其广泛应用。由于埃博霉素的细胞毒性对其产生菌进行转录调控有利于其高效生物合成。本项目将外源功能基因vgb和epoF基因导入至纤维堆囊菌So ce M4中，vgb基因的导入能使该菌株埃博霉素B的产量提高57.9±0.31%，重组质粒pET22b-epoF的导入能使埃博霉素B的产量提高62.7±0.80%，两者同时导入能使埃博霉素B的产量提高122.4±0.70%。本项目扩增获得埃博霉素生物合成基因启动子P3，并验证其功能；将P3启动子核心序列采用生物素进行标记，从So ce M4总蛋白中所得蛋白为So ce M4中新型内源性调控蛋白。将重组TALE-TF载体和dcas9-VP64载体电转化至SoceM4，埃博霉素生物合成基因表达水平显著提升。RP3-TALE-VP64的导入能使埃博霉素B的产量提升2.89倍。dCas9-VP64的导入SoceM4能使埃博霉素B产量提高1.53倍。将RP3-TALE-VP64导入至BL21（DE3），用链霉亲和素磁珠钓取获得纯的RP3-TALE-VP64蛋白。将生物素标记的P3启动子探针与获得的38k Da野生调控蛋白和RP3-TALE-VP64蛋白分别进行相互作用进行EMSA实验，获得了阻滞条带，证明野生调控蛋白和RP3-TALE-VP64蛋白均能和P3启动子相互作用，且两者存在竞争性抑制作用。野生调控蛋白和重组RP3-TALE-VP64的结合区域位于P3启动子的核心区域。本项目还采用TALEN技术对纤维堆囊菌埃博霉素生物合成基因epoK进行了敲除，使埃博霉素D产量提高了66.7%。本项目初步揭示了TALE-TF元件调控纤维堆囊菌埃博霉素生物合成的机制，为埃博霉素的高效生物合成奠定了基础，从而促进埃博霉素的广泛应用。