解淀粉芽孢杆菌中非编码RNA FenS对丰原素合成酶基因表达调控的研究

Fengycin，as a antimicrobial peptide, is produced by Bacillus spp and displays potent antifungal activity against filamentous fungi. Therefore, it exhibits potential application in food preservation and other fields. At present, the production of Fengycin is quite low through fermentation, and the molecular mechanism of fengycin synthesis is still unclear; furthermore, the study about non-coding RNA involved in the post transcriptional regulation of genes has not been reported. Therefore, the feasibility of genetic modification in strain to increase fengycin production is limited due to the lack of the information involved in expression and regulation mechanism of fengycin synthetase gene. In view of this scientific issue, Bacillus amyloliquefaciens is taken as a research object, and the mechanism of interaction between non-coding RNA FenS and gene of fengycin synthetase is going to be investigated in this project. The investigation of the effect of FenS on fengycin synthesis will be performed through knockout and overexpression of FenS gene which is targeting regulatory relations with fengycin synthase gene. Furthermore, the proteomic analysis and site directed mutagenesis will be used to investigate the fengycin synthesis mechanism regulated by sRNA. As a result, the theoretical basis is to be provided for improving fengycin yield by genetic modification of Bacillus amyloliquefaciens.

丰原素是一类由解淀粉芽孢杆菌代谢产生，对丝状真菌具有显著抑菌活性的生物源抗菌肽，在食品防腐等领域具有潜在的应用价值。目前，丰原素产量较低，国内外对丰原素合成的分子调控机理不甚了解，由非编码RNA参与的基因转录后调控更是未见报道。由于缺乏对丰原素合成酶基因表达调控机制的了解，限制了研究人员在分子水平上改造菌株，提高丰原素产量的可行性。针对这一科学问题，本项目以解淀粉芽孢杆菌为研究菌株，对非编码RNA FenS参与丰原素合成酶基因的转录后调控机制进行探讨。通过敲除和过表达与丰原素合成酶基因存在靶向调控关系的非编码RNA FenS，探索FenS对丰原素合成的影响；结合蛋白组学分析及定点突变技术，分析FenS调控fengycin合成的分子机理，为从分子水平上改造解淀粉芽孢杆菌，提高丰原素产量提供理论依据，同时为生物源食品防腐剂的工业化利用奠定基础。

为了揭示解淀粉芽孢杆菌丰原素合成代谢的转录后调控机制，本课题通过对非编码RNA序列sRNA783进行基因序列分析，获得了一段与丰原素合成相关的非编码RNA（FenSr3），该序列位于转录组测序链的正义链上，由355个核苷酸组成。利用穿梭质粒pCBS以及过表达载体pHT43结合同源重组技术，对解淀粉芽孢杆菌FenSr3基因进行敲除和过表达，成功构建了FenSr3基因缺失突变菌株LPB-18N和基因过表达突变菌LPB-18P，结果显示，敲除菌株LPB-18N相比于原始菌株，其Fengycin产量由190.908 mg/L可增加到327.598 mg/L，提高72％。过表达菌株LPB-18P Fengycin产量由190.464 mg/L减少到38.6 mg/L。通过对非编码RNA FenSr3与靶标半衰期分析，证明了非编码RNA FenSr3在解淀粉芽孢杆菌中的负调控功能。利用大分子互作技术（MST）分析了非编码RNA FenSr3基因与靶基因的互作关系，证明了FenSr3与靶mRNA上的作用位点的结合趋势，揭示了FenSr3能够分别与Fengycin合成酶基因中的两个靶mRNA通过碱基互补配对作用结合。通过对解淀粉芽孢杆菌LPB-18N和原始菌株LPB-18进行转录组学和蛋白质组学分析，共筛选得到了1673个差异显著表达的基因，其中显著上调差异基因有702个，显著下调差异基因有971个，这些差异显著基因主要集中在物质代谢，糖酵解，脂肪酸代谢，氨基酸代谢和基因转录调控等过程中。相关研究主要从转录后水平研究解淀粉芽孢杆菌LPB-18 fengycin合成代谢调控机理，为从分子水平改良解淀粉芽孢杆菌，提高的fengycin合成代谢提供理论依据。本研究验证了非编码RNA FenSr3在解淀粉芽孢杆菌LPB-18 fengycin合成代谢中的分子功能，获得了非编码RNA与靶基因mRNA主要通过剪辑互补配对作用结合的作用机制，在本项目的资助下，课题组发表SCI论文3篇，录用中文核心期刊论文1篇，申请发明专利1件，授权发明专利1件。