表观遗传调控哈茨木霉中“沉默”化合物的激活及活性评价

Fungal secondary metabolites (SMs) are important resources for drug discovery and development process. However, it is becoming more and more difficult to identify novel SMs from fungi nowadays. This project focuses on the important biocontrol fungus, Trichoderma harzianum (T. harzianum). Epigenetic regulation strategy was introduced to activate the “silent gene” in this strain in order to obtain new leading compounds. This project will contain the following 4 parts: 1) Identify the epigenetic regulators in T. harzianum by running BLAST with the known ones in model fungi and then construct the gene knock-out or overexpression mutants; 2) Search and isolate the “activated” compounds in the mutated strains compared to WT and elucidate their structures. Calculate the difference of important SMs’ product yields between mutated and WT strains; 3) Test the bioactivity of obtained compounds against fungi and bacteria, etc.; 4) Propose the mechanism of epigenetic regulation in T. harzianum based on RNA seq data and reported literatures. .This project could not only deepen our knowledge about the chemical profile of T. harzianum, but also discovery new SMs with high efficiency. Meanwhile, the mechanism study could be of great help for understanding the physiological and ecological functions of these SMs as well as rational drug discovery.

真菌次级代谢产物是药物的重要来源。然而，随着真菌天然产物化学研究的深入，现有常规技术手段越来越难以发现新的化合物。本项目拟以具有重要生物防治功能的哈茨木霉为研究材料，通过表观遗传调控激活哈茨木霉中编码次级代谢产物的“沉默”基因，获得新的药物先导化合物。具体包括以下内容：1）以模式真菌中调控次级代谢产物的重要表观遗传调控因子为靶标，在哈茨木霉中鉴定这些调控因子，利用基因敲除或过表达技术获得突变株；2）与野生型菌株次级代谢谱比较，寻找突变株中激活的“沉默”产物，鉴定其结构，并对产量发生变化的化合物进行定量分析；3）对分离到的次级代谢产物进行抗菌等生物活性评价；4）通过转录组分析和文献调研，初步探究调控机制。该研究将深化对哈茨木霉化学背景的认识，加速其中天然产物的发现。同时，探索次级代谢产物调控的分子机制，有助于了解相关化合物的生理和生态学功能，从而有利于药物的理性发现。

本项目以具有重要生物防治功能的哈茨木霉为研究材料，通过表观遗传调控激活哈茨木霉中编码次级代谢产物的“沉默”基因，获得新的活性天然产物。主要取得了以下研究进展：1）通过生物信息学分析和RT-PCR，锁定了哈茨木霉基因组中6个表观调控因子；构建了适配于哈茨木霉的CRISPR/Cas9基因编辑系统，获得了7株表观调控因子缺失或过表达突变株。在此过程中发现海盐能够显著诱导多种真菌对潮霉素B的耐受性，并基于转录组数据和透射电镜观察结果，提出三种可能的诱导机制，为真菌耐药机制研究提供案例；2）基于OSMAC策略筛选发现两株突变株与野生型代谢产物存在显著差异。靶向差异代谢产物分离得到7个化合物，其中新化合物3个。分离得到的已知化合物中包括哈茨木霉中微量、需诱导产生的homodimericin A和全合成得到的8-epi-homdimericin A。该工作显著提高了已知化合物（8-epi-）homodimericin A的产量，为其获取提供了一种高效的方法；3）对分离到的次级代谢产物进行细胞毒、抗炎和抗菌等生物活性评价：新化合物哈茨木霉酸A (trichoharzianin A)无细胞毒活性，且具有良好的抗炎活性、8-epi-homodimericin A显示出中等抗植物病原菌活性；4）基于ThpacC敲除和过表达突变株与野生型菌株次级代谢谱和生防活性比较，揭示了ThPacC是homodimericin A生物合成和哈茨木霉生防作用的正调控因子。转录组分析和文献调研，初步解析了ThPacC对homodimericin A生物合成和哈茨木霉生防作用的调控机制，有助于了解次级代谢产物的生理和生态学功能，为抗植物病原菌先导分子的理性发现提供案例。