组蛋白乙酰化表观修饰对黑曲霉次级代谢基因簇活性的差异调控机制

Histone epigenetic modification is an important route to regulate microbial secondary metabolism, which is very important for exploring natural product resources. Preliminary study showed that deletion of histone acetyltransferase GcnE in Aspergillus niger, exhibited discrepancy in regulating the activity of different A. niger secondary metabolite (SM) gene clusters (activation or repression). However, the mechanism for GcnE-mediated regulational discrepancy in A. niger secondary metabolism remains unknown. In this study, LC/MS and NMR techniques will be used to characterize the quantity and structures of the newly synthesized secondary metabolites in A. niger GcnE mutants (overexpression mutant and deletion mutant, etc). And the gene clusters for synthesizing these compounds could be identified via gene knockout. Therefore, we could establish the link between the newly produced compounds and their biosynthetic gene clusters in GcnE mutants. This is the first issue we want to address in this study. Then, ATAC-seq and CHIP-seq techniques are used to detect the difference on the transcriptional activity and the histone epigenetic modification mode (acetylation and methylation) of SM gene clusters in GcnE mutants. So we could investigate the relationship between the transcription activity and the epigenetic modification of GcnE-regulated SM gene clusters, and interpret the mechanism on differentially regulating the expression activity of different A. niger SM gene clusters by epigenetic modification. This is the second issue we want to address in this study. Based on the above studies, CRISPR/nCas9 technique could be used for site-specific regulation of A. niger SM gene clusters by epigenetic regulators, to verify the mechanism on differentially regulating the expression activity of different A. niger SM gene clusters by epigenetic modification, and to activate the biosynthesis of abundant SM compounds, which could provide theoretical and technical foundations for drug exploration using A. niger natural bioactive compounds.

组蛋白表观遗传修饰，是调控微生物次级代谢的重要方法，对开发天然产物资源具有重要意义。前期研究发现，敲除组蛋白乙酰化酶GcnE，表现出对黑曲霉不同次级代谢基因簇活性的调控差异（激活或抑制），其机制尚不清楚。本研究利用LC/MS、NMR技术对GcnE突变株（过表达、敲除等）中新代谢产物的结构和产量，进行定性、定量鉴定，并通过基因敲除验证新产物的合成基因簇，建立代谢产物与合成基因簇的关联。同时，利用ATAC-seq、CHIP-seq技术，检测GcnE突变株中次级代谢基因簇的转录活性及组蛋白表观遗传修饰模式（乙酰化、甲基化）的差异，研究基因簇活性与表观遗传修饰的关系，探讨表观遗传修饰对不同基因簇活性的差异调控机制。在此基础上，利用CRISPR/nCas9技术介导表观遗传因子，特异性定点调控基因簇活性，验证表观遗传修饰的差异调控机制并激活产物的合成，为基于天然活性物质的药物研发提供理论和技术支撑。

本项目针对表观遗传修饰对黑曲霉不同基因簇活性的差异调控机制的关键问题，利用LC/MS、NMR、转录组测序、ATAC-seq等技术，完成了以下研究工作：（1）完成了黑曲霉GcnE突变株的构建及转录谱、代谢产物谱分析，基于LC-MS、NMR技术解析了GcnE突变株中新合成的次级代谢产物的结构。（2）对分离到的6个黑曲霉天然产物进行了NMR结构分析，并对首次在黑曲霉中分离到的新化合物nigerpyrone及其同系物pestalamide A的生物合成途径及基因簇进行了预测和功能验证。（3）利用转录组测序技术研究了黑曲霉组蛋白去乙酰化酶对次级代谢产物合成的影响，并利用ATAC-seq技术研究了GcnE突变株次级代谢基因簇组蛋白乙酰化修饰与染色质开放性、基因表达水平之间的关系。（4）构建了基于CRISPR/dCas9技术的黑曲霉次级代谢基因簇特异性定点表观遗传修饰体系，利用组蛋白乙酰化酶P300和GcnE等，实现了对次级代谢产物brevianamide F、fwnA等的合成基因簇的特异性定点表观遗传调控。以上工作为激活黑曲霉丰富的次级代谢基因簇活性、合成更多的活性天然产物奠定了基础，为基于活性天然产物的药物研发提供了理论和技术支撑。